Preparation of 7-dehydrocholesterol and/or the biosynthetic intermediates and/or secondary products thereof in transgenic organisms

ABSTRACT

The present invention relates to a method for preparing 7-dehydrocholesterol and/or the biosynthetic intermediates and/or secondary products thereof by culturing organisms, in particular yeasts. Furthermore, the invention relates to the preparation of the nucleic acid constructs required for preparing the genetically modified organisms and to said genetically modified organisms, in particular yeasts, themselves.

The present invention relates to a method for preparing7-dehydrocholesterol and/or the biosynthetic intermediates and/orsecondary products thereof by culturing organisms, in particular yeasts.Furthermore, the invention relates to the preparation of the nucleicacid constructs required for preparing the genetically modifiedorganisms and to said genetically modified organisms, in particularyeasts, themselves.

7-Dehydrocholesterol, also referred to as cholesta-5,7-dienol orprovitamin D3, its biosynthetic intermediates of the sterol metabolism,such as, for example, zymosterol, farnesol, geraniol, squalene,lanosterol, cholesta-5,7,24-trienol and cholesta-5,7,22,24-tetraenol andits biosynthetic secondary products of the sterol metabolism, such asvitamin D₃ and cholesterol, are compounds of high economic value.

7-Dehydrocholesterol is economically important especially for obtainingvitamin D₃ from 7-dehydrocholesterol via UV irradiation.

Squalene is used as building block for the synthesis of terpenes. It isused in hydrogenated form as squalane in dermatology and cosmetics andalso in various derivatives as an ingredient of skin and haircareproducts.

Furthermore, sterols such as zymosterol and lanosterol can be utilizedeconomically, lanosterol being pivotal as crude and synthesis materialfor the chemical synthesis of saponins and steroid hormones. Due to itsgood skin penetration and spreading properties, lanosterol serves asemulsifier and active substance in skin creams.

An economic method for preparing 7-dehydrocholesterol and/or thebiosynthetic intermediates and/or secondary products thereof istherefore of great importance.

Particularly economic methods are biotechnological methods utilizingorganisms which have been optimized by genetic modification and whichproduce 7-dehydrocholesterol and/or the biosynthetic intermediatesand/or secondary products thereof.

While the sterol metabolism in bacteria, fungi, yeasts and some insectsessentially goes from zymosterol via fecosterol, episterol,ergosta-5,7-dienol and ergosta-5,7,22,24-tetraen-3β-ol to ergosterol(provitamin D₂), the sterol metabolism in mammals essentially goes fromzymosterol via cholesta-7,24-dienol, lathosterol to 7-dehydrocholesterol(provitamin D₃).

7-Dehydrocholesterol (provitamin D₃) is converted to cholesterol by7-dehydrocholesterol reductase and cholesterol is converted to steroidhormones, corticoids and bile acids, such as progesterone, testosterone,estradiol, aldosterone, cortisone and cholate.

Some genes of the 7-dehydrocholesterol metabolism in mammals are knownand have been cloned, such as, for example, nucleic acids encoding ahuman Δ8-Δ7-isomerase (also referred to as emopamil-binding protein(EBP)), ACCESSION NM_(—)006579, and a murine Δ8-Δ7-isomerase (Braverman,N. et al., (1999): Mutations in the gene encoding3β-hydroxysteroid-Δ8,Δ7-isomerase cause X-linked dominantConradi-Hunermann syndrome. Nat. Genet. 22(3), 291-294),

nucleic acids encoding a human 45-desaturase (also referred to as sterolC5-desaturase), ACCESSION AB016247 and a murine Δ5-desaturase (Nishi, S.et al., (2000): cDNA cloning of the mammalian sterol C5-desaturase andthe expression in yeast mutant. Biochim. Biophys. Acta 1490(1-2),106-108),

nucleic acids encoding a human Δ24-reductase (also referred to as24-dehydrocholesterol reductase (DHCR24)), ACCESSION NM_(—)014762 and amurine Δ24-reductase (Waterham, H. R. et al. (2001): Mutations in the3β-hydroxysterol Δ24-reductase gene cause desmosterolosis, an autosomalrecessive disorder of cholesterol biosynthesis. Am. J. Hum. Genet.69(4), 685-694) and

nucleic acids encoding a human sterol acyltransferase (Chang, C. C. etal., Molecular cloning and functional expression of human acyl-coenzymeA:cholesterol acyltransferase cDNA in mutant Chinese hamster ovarycells, J. Biol. Chem. 1993, Oct. 5; 268(28):20747-55) and a murinesterol acyltransferase (Uelmen, P. J.: Tissue-specific expression andcholesterol regulation of acylcoenzyme A:cholesterol acyltransferase(ACAT) in mice. Molecular cloning of mouse ACAT cDNA, chromosomallocalization, and regulation of ACAT in vivo and in vitro, J. Biol.Chem. 1995 Nov. 3; 270(44):26192-201).

The genes of the ergosterol metabolism in yeast are essentially knownand have been cloned, such as, for example,

nucleic acids encoding a Δ8-Δ7-isomerase (ERG2) (Ashman, W. H. et al.(1991): Cloning and disruption of the yeast C-8 sterol isomerase gene.Lipids. August; 26(8):628-32.),

Nucleic acids encoding a 45-desaturase (ERG3) (Arthington, B. A. et al.(1991): Cloning, disruption and sequence of the gene encoding yeast C-5sterol desaturase. Gene. June 15; 102(1):39-44.),

nucleic acids encoding a Δ24-reductase (ERG 4) (Lai, M. H. et al.,(1994): The identification of a gene family in the Saccharomycescerevisiae ergosterol biosynthesis pathway. Gene. March 11;140(1):41-9.),

nucleic acids encoding an HMG-CoA reductase (HMG)(Bason M. E. et al,(1988) Structural and functional conservation between yeast and human3-hydroxy-3-methylglutaryl coenzyme A reductases, the rate-limitingenzyme of sterol biosynthesis. Mol Cell Biol 8:3797-3808,

nucleic acids encoding a truncated HMG-CoA reductase (t-HMG) (PolakowskiT, Stahl U, Lang C. (1998) Overexpression of a cytosolichydroxymethylglutaryl-CoA reductase leads to squalene accumulation inyeast. Appl Microbiol Biotechnol. January; 49(1):66-71,

nucleic acids encoding a lanosterol C14-demethylase (ERG11) (Kalb V F,Loper J C, Dey C R, Woods C W, Sutter T R (1986) Isolation of acytochrome P-450 structural gene from Saccharomyces cerevisiae. Gene45(3):237-45,

nucleic acids encoding a squalene synthetase (ERG9) (Jennings, S. M.,(1991): Molecular cloning and characterization of the yeast gene forsqualene synthetase. Proc Natl Acad Sci USA. July 15; 88(14):6038-42),

nucleic acids encoding a sterol acyltransferase (SAT1) and (SAT2) (Yang,H.: Sterol esterification in yeast: a two-gene process. Science. 1996May 31; 272(5266):1353-6.) and a further sterol acyltransferase (J.Biol. Chem. 1996, Sep. 27; 271(39):24157-63), nucleic acids encoding asqualene epoxidase (ERG1) (Jandrositz, A., et al (1991) The geneencoding squalene epoxidase from Saccharomyces cerevisiae: cloning andcharacterization. Gene 107:155-160),

nucleic acids encoding a C24-methyltransferase (ERG6) (Hardwick, K. G.et al.: SED6 is identical to ERG6, and encodes a putativemethyltransferase required for ergosterol synthesis. Yeast. February;10(2):265-9) and

nucleic acids encoding a Δ22-desaturase (ERG5) (Skaggs, B. A. et al.:Cloning and characterization of the Saccharomyces cerevisiae C-22 steroldesaturase gene, encoding a second cytochrome P-450 involved inergosterol biosynthesis, Gene. 1996 Feb. 22; 169(1):105-9).

Furthermore, methods are known whose aim is an increase in the contentof specific intermediates and final products of the sterol metabolism inyeasts and fungi.

EP 486 290 discloses that the content of squalene and other specificsterols such as, for example, zymosterol, in yeasts can be increased byincreasing the rate of expression of HMG-CoA reductase andsimultaneously interrupting the metabolic pathway of zymosterolC24-methyltransferase (ERG6) and ergosta-5,7,24(28)-trienol22-dehydrogenase (ERG5).

From T. Polakowski, Molekularbiologische Beeinflussung desErgosterolstoffwechsels der Hefe Saccharomyces cerevisiae [Influencingthe ergosterol metabolism of the yeast Saccharomyces cerevisiae bymolecular biological means], Shaker Verlag Aachen, 1999, pages 59 to 66,it is known that increasing the rate of expression of HMG-CoA reductasealone, without interrupting the downstream metabolic flow as in EP 486290, merely leads to a slight increase in the content of early sterolsand of squalene, while the content of later sterols such as ergosteroldoes not change substantially and, in the case of ergosterol, eventendentially decreases.

WO 99/16886 describes a method for preparing ergosterol in yeasts whichoverexpress a combination of genes tHMG, ERG9, SAT1 and ERG1.

Tainaka et al., J, Ferment. Bioeng. 1995, 79, 64-66, further describethat overexpression of ERG11 (lanosterol C14-demethylase) leads toaccumulation of 4,4-dimethylzymosterol but not of ergosterol. Comparedto the wild type, the transformant showed an increase in the zymosterolcontent by a factor of from 1.1 to 1.47, depending on fermentationconditions.

Avruch et al, Can. J. Biochem 1976, 54(7), 657-665 and Xu et al,Biochem. Biophys. Res. Commun 1988, 155(1), 509-517 describe that it ispossible to detect, apart from zymosterol, also traces of cholesterol byspecifically inhibiting C24-methyltransferase and also by a mutation inthe gene locus erg6 in S. cerevisiae.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the vector denoted pUG6-tHMG.

FIG. 2 illustrates the vector denoted pUG6-ERG1.

FIG. 3 illustrates the vector denoted pUG6-ERG11.

FIG. 4 illustrates the vectors denoted pFlat3 and pFlat1.

FIG. 5 a illustrates the vectors denoted pFlat1-EBP and pFlat3-EBP.

FIG. 5 b illustrates the vector denoted pFlat3-SC5D.

FIG. 5 c illustrates the vector denoted pFlat1-Ebp.

FIG. 5 d illustrates the vector denoted pFlat4-D24R.

FIG. 6 illustrates the vector denoted pFlat4.

FIG. 7 illustrates the vector denoted pFlat4-ERG4.

It is an object of the present invention to provide a method forpreparing 7-dehydrocholesterol and/or the biosynthetic intermediatesand/or secondary products thereof, which method has advantageousproperties such as a higher product yield.

We have found that this object is achieved by a method for preparing7-dehydrocholesterol and/or the biosynthetic intermediates and/orsecondary products thereof, in which organisms are cultured which have,compared to the wild type, an increased activity of at least one of theactivities selected from the group consisting of Δ8-Δ7-isomeraseactivity, Δ5-desaturase activity and Δ24-reductase activity.

An increased activity compared to the wild type means, in the case ofthe starting organism not having said activity, that said activity iscaused. In the case of the starting organism already having saidactivity, an increased activity compared to the wild type means anactivity increased by a percentage.

Δ8-Δ7-Isomerase activity means the enzyme activity of a Δ8-Δ7-isomerase,also referred to as Δ8-Δ7-sterol isomerase.

A Δ8-Δ7-isomerase means a protein which has the enzyme activity ofconverting zymosterol to cholesta-7,24-dienol.

Accordingly, Δ8-Δ7-isomerase activity means the amount of zymosterolconverted or the amount of cholesta-7,24-dienol formed by the proteinΔ8-Δ7-isomerase in a particular time.

In the case of an increased Δ8-Δ7-isomerase activity compared to thewild type, thus the amount of zymosterol converted or the amount ofcholesta-7,24-dienol formed by the protein Δ8-Δ7-isomerase in aparticular time is increased in comparison with the wild type.

This increase in Δ8-Δ7-isomerase activity is preferably at least 5%,further preferably at least 20%, further preferably at least 50%,further preferably at least 100%, more preferably at least 300%, stillmore preferably at least 500%, in particular at least 600%, of theΔ8-Δ7-isomerase activity of the wild type.

Δ5-Desaturase activity means the enzyme activity of a Δ5-desaturase,also referred to as lathosterol 5-desaturase or sterol C5-desaturase.

A Δ5-desaturase means a protein which has the enzyme activity ofconverting cholesta-7,24-dienol to cholesta-5,7,24-trienol.

Accordingly, Δ5-desaturase activity means the amount ofcholesta-7,24-dienol converted or the amount of cholesta-5,7,24-trienolformed by the protein Δ5-desaturase in a particular time.

In the case of an increased Δ5-desaturase activity compared to the wildtype, thus the amount of cholesta-7,24-dienol converted or the amount ofcholesta-5,7,24-trienol formed by the protein Δ5-desaturase in aparticular time is increased in comparison with the wild type.

This increase in Δ5-desaturase activity is preferably at least 5%,further preferably at least 20%, further preferably at least 50%,further preferably at least 100%, more preferably at least 300%, stillmore preferably at least 500%, in particular at least 600%, of theΔ5-desaturase activity of the wild type.

Δ24-Reductase activity means the enzyme activity of a Δ24-reductase,also referred to as 24-dehydrocholesterol reductase.

A Δ24-reductase means a protein which has the enzyme activity ofconverting the double bond between C24 and C25 of cholesterol compoundsto a single bond, for example converting cholesta-5,7,24-trienol to7-dehydrocholesterol or zymosterol to lathosterol orcholesta-7,24-dienol to cholesta-7-enol.

Accordingly, Δ24-reductase activity means preferably the amount ofcholesta-5,7,24-trienol converted or the amount of 7-dehydrocholesterolformed by the protein Δ24-reductase in a particular time.

In the case of an increased Δ24-reductase activity compared to the wildtype, thus the amount of cholesta-5,7,24-trienol converted or the amountof 7-dehydrocholesterol formed by the protein Δ24-reductase in aparticular time is increased in comparison with the wild type.

This increase in Δ24-reductase activity is preferably at least 5%,further preferably at least 20%, further preferably at least 50%,further preferably at least 100%, more preferably at least 300%, stillmore preferably at least 500%, in particular at least 600%, of theΔ24-reductase activity of the wild type.

A wild type means the corresponding not genetically modified startingorganism. Preferably and, in particular in those cases in which theorganism or the wild type cannot be classified unambiguously, wild typemeans a reference organism for increasing the Δ8-Δ7-isomerase activity,increasing the Δ5-desaturase activity, increasing the Δ24-reductaseactivity, reducing the C24-methyltransferase activity described below,reducing the Δ22-desaturase activity described below, increasing theHMG-CoA-reductase activity described below, increasing the lanosterolC14-demethylase activity described below, increasing thesqualene-epoxidase activity described below, increasing thesqualene-synthetase activity described below and increasing thesterol-acyltransferase activity described below and also for increasingthe content of 7-dehydrocholesterol and/or of the biosyntheticintermediates and/or secondary products thereof. This reference organismis preferably the yeast strain Saccharomyces cerevisiae AH22.

In the method of the invention, organisms are cultured which, comparedto the wild type, have an increased activity of at least one of theactivities selected from the group consisting of Δ8-Δ7-isomeraseactivity, Δ5-desaturase activity and Δ24-reductase activity.

In a preferred embodiment, organisms are cultured which, compared to thewild type, have an increased Δ8-Δ7-isomerase activity, Δ5-desaturaseactivity or Δ24-reductase activity.

In a particularly preferred embodiment of the method of the invention,the organisms have, compared to the wild type, an increased activity ofat least two of the activities selected from the group consisting ofΔ8-Δ7-isomerase activity, Δ5-desaturase activity and Δ24-reductaseactivity.

Particularly preferred combinations are Δ8-Δ7-isomerase activity andΔ5-desaturase activity, increased in comparison to the wild type,Δ8-Δ7-isomerase activity and Δ24-reductase activity, increased incomparison to the wild type, and Δ5-desaturase activity andΔ24-reductase activity, increased in comparison with the wild type.

In a very particularly preferred embodiment of the method of theinvention, the organisms have, compared to the wild type, an increasedΔ8-Δ7-isomerase activity, Δ5-desaturase activity and Δ24-reductaseactivity.

The Δ8-Δ7-isomerase activity, Δ5-desaturase activity and Δ24-reductaseactivity and also the HMG-CoA-reductase activity, lanosterolC14-demethylase activity, squalene-epoxidase activity,squalene-synthetase activity and sterol-acyltransferase activity, whichare described below, may be increased independently of one another invarious ways, for example by eliminating inhibiting regulatorymechanisms at the expression and protein level or by increasing,compared to the wild type, gene expression of the corresponding nucleicacids, i.e. nucleic acids encoding a Δ8-Δ7-isomerase, Δ5-desaturase,Δ24-reductase, HMG-CoA reductase, lanosterol C14-demethylase, squaleneepoxidase, squalene synthetase or sterol acyltransferase.

Likewise, gene expression of the corresponding nucleic acid may beincreased compared to the wild type in various ways, for example byinducing the appropriate genes by activators, i.e. by inducing theΔ8-Δ7-isomerase gene, the Δ5-desaturase gene, the Δ24-reductase gene,the HMG-CoA-reductase gene, the lanosterol C14-demethylase gene, thesqualene-epoxidase gene, the squalene-synthetase gene or thesterol-acyltransferase gene by activators, or by introducing one or moregene copies of the appropriate nucleic acids, i.e. by introducing one ormore nucleic acids encoding a Δ8-Δ7-isomerase, Δ5-desaturase,Δ24-reductase, HMG-CoA reductase, lanosterol C14-demethylase, squaleneepoxidase, squalene synthetase or sterol acyltransferase into theorganism.

Increasing the gene expression of a nucleic acid encoding aΔ8-Δ7-isomerase, Δ5-desaturase, Δ24-reductase, HMG-CoA reductase,lanosterol C14-demethylase, squalene epoxidase, squalene synthetase orsterol acyltransferase means according to the invention alsomanipulation of the expression of endogenous Δ8-Δ7-isomerases,Δ5-desaturases, Δ24-reductases, HMG-CoA reductases, lanosterolC14-demethylases, squalene epoxidases, squalene synthetases or sterolacyltransferases, which are intrinsic to the organism, in particular tothe yeasts.

This may be achieved, for example, by modifying the promoter DNAsequence of genes coding for Δ8-Δ7-isomerase, Δ5-desaturase,Δ24-reductase, HMG-CoA reductase, lanosterol C14-demethylase, squaleneepoxidase, squalene synthetase or sterol acyltransferase. Such amodification which causes an increased rate of expression of therelevant gene may be carried out, for example, by deleting or insertingDNA sequences.

As described above, it is possible to modify expression of theendogenous, Δ8-Δ7-isomerase, Δ5-desaturase, Δ24-reductase, HMG-CoAreductase, lanosterol C14-demethylase, squalene epoxidase, squalenesynthetase or sterol acyltransferase by applying exogenous stimuli. Thismay be carried out using particular physiological conditions, i.e. byapplying foreign substances.

Furthermore, a modified or increased expression of endogenousΔ8-Δ7-isomerase, Δ5-desaturase, Δ24-reductase, HMG-CoA reductase,lanosterol C14-demethylase, squalene epoxidase, squalene synthetase orsterol acyltransferase genes may be achieved by interaction of aregulatory protein which is not present in the untransformed organismwith the promoter of said genes.

A regulator of this type may be a chimeric protein which consists of aDNA-binding domain and a transcriptional activator domain, as described,for example, in WO 96/06166.

In a preferred embodiment, the Δ8-Δ7-isomerase activity is increasedcompared to the wild type by increasing the gene expression of a nucleicacid encoding a Δ8-Δ7-isomerase.

In a further preferred embodiment, gene expression of a nucleic acidencoding a Δ8-Δ7-isomerase is increased by introducing into the organismone or more nucleic acids encoding a Δ8-Δ7-isomerase.

For this purpose, it is possible to use in principle any Δ8-Δ7-isomerasegene, i.e. any nucleic acid encoding a Δ8-Δ7-isomerase.

In the case of genomic Δ8-Δ7-isomerase nucleic acid sequences fromeukaryotic sources, which contain introns, preferably already processednucleic acid sequences such as the corresponding cDNAs are to be used,if the host organism is unable to or cannot be enabled to express theappropriate Δ8-Δ7-isomerase.

Examples of Δ8-Δ7-isomerase genes are nucleic acids encoding a murineΔ8-Δ7-isomerase (nucleic acid: Seq. ID. No. 1, protein: Seq. ID. No. 2)or a human Δ8-Δ7-isomerase (nucleic acid: Seq. ID. No. 3, protein: Seq.ID. No. 4) (Braverman, N. et al., (1999): Mutations in the gene encoding

3β-hydroxysteroid-Δ8,Δ7-isomerase cause X-linked dominantConradi-Hunermann syndrome, Nat. Genet. 22(3), 291-294), or else nucleicacids encoding proteins which have the activity of a Δ8-Δ7-isomerase,for example due to a broad substrate specificity, such as, for example,nucleic acids encoding a C8-isomerase Saccharomyces cerevisiae (ERG2)(Nucleic acid: Seq. ID. No. 5, protein: Seq. ID. No. 6) (Ashman, W. H.et al. (1991): Cloning and disruption of the yeast C-8 sterol isomerasegene. Lipids. August; 26(8):628-32).

In this preferred embodiment, thus at least one further Δ8-Δ7-isomerasegene is present in the transgenic organisms of the invention, comparedto the wild type.

The number of Δ8-Δ7-isomerase genes in the transgenic organisms of theinvention is at least two, preferably more than two, particularlypreferably more than three and very particularly preferably more thanfive.

All of the nucleic acids mentioned in the description may be, forexample, an RNA sequence, DNA sequence or cDNA sequence.

Preferred Δ8-Δ7-isomerase genes are nucleic acids encoding proteinswhich have a high substrate specificity for zymosterol. Therefore,preference is given in particular to Δ8-Δ7-isomerase genes and to thecorresponding Δ8-Δ7-isomerases of mammals and to the functionalequivalents thereof.

Accordingly, preference is given to using in the above-described methodnucleic acids which encode proteins comprising the amino acid sequenceSEQ. ID. NO. 2 or a sequence derived from this sequence by substitution,insertion or deletion of amino acids, which is at least 30%, preferablyat least 50%, more preferably at least 70%, still more preferably atleast 90%, most preferably at least 95%, identical at the amino acidlevel with the sequence SEQ. ID. NO. 2, and having the enzyme propertyof a Δ8-Δ7-isomerase.

The sequence SEQ. ID. NO. 2 represents the amino acid sequence of Musmusculus Δ8-Δ7-isomerase.

Further examples of Δ8-Δ7-isomerases and Δ8-Δ7-isomerase genes canreadily be found, for example, for various organisms whose genomicsequence is known by comparing the homology of the amino acid sequencesor the corresponding backtranslated nucleic acid sequences fromdatabases with the SeQ ID. NO. 2.

The Homo sapiens Δ8-Δ7-isomerase (Seq. ID. No. 4), for example, is 74%identical to the Mus musculus Δ8-Δ7-isomerase (Seq. ID. No. 2).

Further examples of Δ8-Δ7-isomerases and Δ8-Δ7-isomerase genes canfurthermore readily be found for various organisms whose genomicsequence is unknown, for example starting from the sequence SEQ. ID. No.1, by hybridization techniques and PCR techniques in a manner known perse.

The term “substitution” means in the description the replacement of oneor more amino acids by one or more amino acids. Preference is given tocarrying out “conservative” replacements in which the replacing aminoacid has a similar property to that of the original amino acid, forexample replacement of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile,Ser by Thr.

A deletion is the replacement of an amino acid by a direct bond.Preferred positions for deletions are the polypeptide termini and thelinkages between the individual protein domains.

Insertions are introductions of amino acids into the polypeptide chain,with a direct bond formally being replaced by one or more amino acids.

Identity between two proteins means identity of the amino acids over thein each case entire length of the protein, in particular the identitywhich is calculated by comparison with the aid of the Lasergene softwarefrom DNASTAR Inc., Madison, Wis. (USA), using the Clustal method(Higgins D G, Sharp P M. Fast and sensitive multiple sequence alignmentson a microcomputer. Comput Appl. Biosci. 1989 April; 5(2):151-1) andsetting the following parameters: TABLE-US-00001 Multiple alignmentparameter: Gap penalty 10 Gap length penalty 10 Pairwise alignmentparameter: K-tuple 1 Gap penalty 3 Window 5 Diagonals saved 5

Accordingly, a protein which is at least 30% identical at the amino acidlevel with the sequence SEQ. ID. NO. 2 means a protein which is at least30% identical when comparing its sequence with the sequence SEQ. ID. NO.2, in particular according to the above program algorithm with the aboveset of parameters.

In a further, particularly preferred embodiment, the Δ8-Δ7-isomeraseactivity is increased by introducing into organisms nucleic acids whichencode proteins comprising the amino acid sequence of Mus musculusΔ8-Δ7-isomerase (SEQ. ID. NO. 2).

Suitable nucleic acid sequences can be obtained, for example, bybacktranslating the polypeptide sequence according to the genetic code.

Preference is given to using for this those codons which are frequentlyused according to the organism-specific codon usage. Said codon usagecan readily be determined on the basis of computer analyses of otherknown genes of the organisms in question.

If the protein is to be expressed in yeast, for example, it is oftenadvantageous to use the codon usage of yeast for backtranslation.

In a particularly preferred embodiment, a nucleic acid comprising thesequence SEQ. ID. NO. 1 is introduced into the organism.

The sequence SEQ. ID. NO. 1 represents the Mus musculus cDNA whichencodes the Δ8-Δ7-isomerase of the sequence SEQ ID NO. 2.

Furthermore, all of the Δ8-Δ7-isomerase genes mentioned above can beprepared in a manner known per se by chemical synthesis from thenucleotide building blocks, for example by fragment condensation ofindividual overlapping complementary nucleic acid building blocks of thedouble helix. The chemical synthesis of oligonucleotides may be carriedout, for example, in a known manner according to the phosphoramiditemethod (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).Annealing of synthetic oligonucleotides and filling-in of gaps with theaid of the Klenow fragment of DNA polymerase and the ligation reactionsand also general cloning methods are described in Sambrook et al.(1989), Molecular cloning: A laboratory manual, Cold Spring HarborLaboratory Press.

In a preferred embodiment, the Δ5-desaturase activity is increasedcompared to the wild type by increasing the gene expression of a nucleicacid encoding a Δ5-desaturase.

In a further preferred embodiment, gene expression of a nucleic acidencoding a Δ5-desaturase is increased by introducing into the organismone or more nucleic acids encoding a Δ5-desaturase.

For this purpose, it is possible to use in principle any Δ5-desaturasegene, i.e. any nucleic acid encoding a Δ5-desaturase.

In the case of genomic Δ5-desaturase nucleic acid sequences fromeukaryotic sources, which contain introns, preferably already processednucleic acid sequences such as the corresponding cDNAs are to be used,if the host organism is unable to or cannot be enabled to express theappropriate Δ5-desaturase.

Examples of Δ5-desaturase genes are nucleic acids encoding a murineΔ5-desaturase (nucleic acid: Seq. ID. No. 7, protein: Seq. ID. No. 8) ora human Δ5-desaturase (nucleic acid: Seq. ID. No. 9, protein: Seq. ID.No. 10) (Nishi, S. et al., (2000): cDNA cloning of the mammalian sterolC5-desaturase and the expression in yeast mutant. Biochim. Biophys.Acta, 1490, (1-2), 106-108), or else nucleic acids encoding proteinswhich have the activity of a Δ5-desaturase, for example due to a broadsubstrate specificity, such as, for example, nucleic acids encoding aSaccharomyces cerevisiae C5-desaturase (ERG3) (nucleic acid: Seq. ID.No. 11, protein: Seq. ID. No. 12), (Arthington, B. A. et al. (1991):Cloning, disruption and sequence of the gene encoding yeast C-5 steroldesaturase. Gene. June 15; 102(1):39-44.).

In this preferred embodiment, thus at least one further Δ5-desaturasegene is present in the transgenic organisms of the invention, comparedto the wild type.

The number of Δ5-desaturase genes in the transgenic organisms of theinvention is at least two, preferably more than two, particularlypreferably more than three and very particularly preferably more thanfive.

Preferred Δ5-desaturase genes are nucleic acids encoding proteins whichhave a high substrate specificity for cholesta-7,24-dienol. Therefore,preference is given in particular to Δ5-desaturase genes and to thecorresponding Δ5-desaturases of mammals and to the functionalequivalents thereof.

Accordingly, preference is given to using in the above-described methodnucleic acids which encode proteins comprising the amino acid sequenceSEQ. ID. NO. 8 or a sequence derived from this sequence by substitution,insertion or deletion of amino acids, which is at least 30%, preferablyat least 50%, more preferably at least 70%, still more preferably atleast 90%, most preferably at least 95%, identical at the amino acidlevel with the sequence SEQ. ID. NO. 8, and having the enzyme propertyof a Δ5-desaturase.

The sequence SEQ. ID. NO. 8 represents the amino acid sequence of Musmusculus Δ5-desaturase.

Further examples of Δ5-desaturase and Δ5-desaturase genes can readily befound, for example, for various organisms whose genomic sequence isknown by comparing the homology of the amino acid sequences or thecorresponding backtranslated nucleic acid sequences from databases withthe SeQ ID. NO. 2.

The Homo sapiens Δ5-desaturase (Seq. ID. No. 10), for example, is 84%identical to Mus musculus Δ5-desaturase (Seq. ID. No. 8).

Further examples of Δ5-desaturases and Δ5-desaturase genes canfurthermore readily be found for various organisms whose genomicsequence is unknown, for example starting from the sequence SEQ. ID. No.7, by hybridization techniques and PCR techniques in a manner known perse.

Accordingly, a protein which is at least 30% identical at the amino acidlevel with the sequence SEQ. ID. NO. 8 means a protein which is at least30% identical when comparing its sequence with the sequence SEQ. ID. NO.8, in particular according to the above program algorithm with the aboveset of parameters.

In a further, particularly preferred embodiment, the Δ5-desaturaseactivity is increased by introducing into organisms nucleic acids whichencode proteins comprising the amino acid sequence of Mus musculusΔ5-desaturase (SEQ. ID. NO. 8).

Suitable nucleic acid sequences can be obtained, for example, bybacktranslating the polypeptide sequence according to the genetic code.

Preference is given to using for this those codons which are frequentlyused according to the organism-specific codon usage. Said codon usagecan readily be determined on the basis of computer analyses of otherknown genes of the organisms in question.

If the protein is to be expressed in yeast, for example, it is oftenadvantageous to use the codon usage of yeast for backtranslation.

In a particularly preferred embodiment, a nucleic acid comprising thesequence SEQ. ID. NO. 7 is introduced into the organism.

The sequence SEQ. ID. NO. 7 represents the Mus musculus cDNA whichencodes the Δ5-desaturase of the sequence SEQ ID NO. 8.

Furthermore, all of the Δ5-desaturase genes mentioned above can beprepared in a manner known per se by chemical synthesis from thenucleotide building blocks, for example by fragment condensation ofindividual overlapping complementary nucleic acid building blocks of thedouble helix. The chemical synthesis of oligonucleotides may be carriedout, for example, in a known manner according to the phosphoramiditemethod (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).Annealing of synthetic oligonucleotides and filling-in of gaps with theaid of the Klenow fragment of DNA polymerase and the ligation reactionsand also general cloning methods are described in Sambrook et al.(1989), Molecular cloning: A laboratory manual, Cold Spring HarborLaboratory Press.

In a preferred embodiment, the Δ24-reductase activity is increasedcompared to the wild type by increasing the gene expression of a nucleicacid encoding a Δ24-reductase.

In a further preferred embodiment, gene expression of a nucleic acidencoding a Δ24-reductase is increased by introducing into the organismone or more nucleic acids encoding a Δ24-reductase.

For this purpose, it is possible to use in principle any Δ24-reductasegene, i.e. any nucleic acid encoding a Δ24-reductase.

In the case of genomic Δ24-reductase nucleic acid sequences fromeukaryotic sources, which contain introns, preferably already processednucleic acid sequences such as the corresponding cDNAs are to be used,if the host organism is unable to or cannot be enabled to express theappropriate Δ24-reductase.

Examples of Δ24-reductase genes are nucleic acids encoding a murineΔ24-reductase (nucleic acid: Seq. ID. No. 13, protein: Seq. ID. No. 14)or a human Δ24-reductase (nucleic acid: Seq. ID. No. 15, protein: Seq.ID. No. 16) (Waterham, H. R. et al.: Mutations in the 3β-HydroxysterolΔ24-Reductase Gene Cause Desmosterolosis, an Autosomal RecessiveDisorder of Cholesterol Biosynthesis, Am. J. Hum. Genet. 69 (4), 685-694(2001)), or else nucleic acids encoding proteins which have the activityof a Δ24-reductase, for example due to a broad substrate specificity,such as, for example, nucleic acids encoding a Saccharomyces cerevisiaeΔ24-reductase (ERG4) (nucleic acid: Seq. ID. No. 17, protein: Seq. ID.No. 18) (Lai, M. H. et al., (1994): The identification of a gene familyin the Saccharomyces cerevisiae ergosterol biosynthesis pathway. Gene.March 11; 140(1):41-9).

In this preferred embodiment, thus at least one further Δ24-reductasegene is present in the transgenic organisms of the invention, comparedto the wild type.

The number of Δ24-reductase genes in the transgenic organisms of theinvention is at least two, preferably more than two, particularlypreferably more than three and very particularly preferably more thanfive.

Preferred Δ24-reductase genes are nucleic acids encoding proteins whichhave a high substrate specificity for cholesta-5,7,24-trienol.Therefore, preference is given in particular to Δ24-reductase genes andto the corresponding Δ24-reductase of mammals and to the functionalequivalents thereof.

Accordingly, preference is given to using in the above-described methodnucleic acids which encode proteins comprising the amino acid sequenceSEQ. ID. NO. 14 or a sequence derived from this sequence bysubstitution, insertion or deletion of amino acids, which is at least30%, preferably at least 50%, more preferably at least 70%, still morepreferably at least 90%, most preferably at least 95%, identical at theamino acid level with the sequence SEQ. ID. NO. 14, and having theenzyme property of a Δ24-reductase.

The sequence SEQ. ID. NO. 14 represents the amino acid sequence of Musmusculus Δ24-reductase.

Further examples of Δ24-reductases and Δ24-reductase genes can readilybe found, for example, for various organisms whose genomic sequence isknown by comparing the homology of the amino acid sequences or thecorresponding backtranslated nucleic acid sequences from databases withthe SeQ ID. NO. 14.

The Homo sapiens Δ24-reductase (Seq. ID. No. 16), for example, is 96%identical to Mus musculus Δ24-reductase (Seq. ID. No. 14).

Further examples of Δ24-reductases and Δ24-reductase genes canfurthermore readily be found for various organisms whose genomicsequence is unknown, for example starting from the sequence SEQ. ID. No.13, by hybridization techniques and PCR techniques in a manner known perse.

Accordingly, a protein which is at least 30% identical at the amino acidlevel with the sequence SEQ. ID. NO. 14 means a protein which is atleast 30% identical when comparing its sequence with the sequence SEQ.ID. NO. 14, in particular according to the above program algorithm withthe above set of parameters.

In a further, particularly preferred embodiment, the Δ24-reductaseactivity is increased by introducing into organisms nucleic acids whichencode proteins comprising the amino acid sequence of Mus musculusΔ24-reductase (SEQ. ID. NO. 14).

Suitable nucleic acid sequences can be obtained, for example, bybacktranslating the polypeptide sequence according to the genetic code.

Preference is given to using for this those codons which are frequentlyused according to the organism-specific codon usage. Said codon usagecan readily be determined on the basis of computer analyses of otherknown genes of the organisms in question.

If the protein is to be expressed in yeast, for example, it is oftenadvantageous to use the codon usage of yeast for backtranslation.

In a particularly preferred embodiment, a nucleic acid comprising thesequence SEQ. ID. NO. 13 is introduced into the organism.

The sequence SEQ. ID. NO. 13 represents the Mus musculus genomic DNAwhich encodes the Δ24-reductase of the sequence SEQ ID NO. 14.

Furthermore, all of the Δ24-reductase genes mentioned above can beprepared in a manner known per se by chemical synthesis from thenucleotide building blocks, for example by fragment condensation ofindividual overlapping complementary nucleic acid building blocks of thedouble helix. The chemical synthesis of oligonucleotides may be carriedout, for example, in a known manner according to the phosphoramiditemethod (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).Annealing of synthetic oligonucleotides and filling-in of gaps with theaid of the Klenow fragment of DNA polymerase and the ligation reactionsand also general cloning methods are described in Sambrook et al.(1989), Molecular cloning: A laboratory manual, Cold Spring HarborLaboratory Press.

In a further preferred embodiment of the method of the invention,organisms are cultured which have, compared to the wild type, a reducedactivity of at least one of the activities selected from the groupconsisting of C24-methyltransferase activity and Δ22-desaturase activityin addition to the above-described genetic modifications.

In a further particularly preferred embodiment, organisms are culturedwhich have, compared to the wild type, a reduced C24-methyltransferaseactivity and a reduced Δ22-desaturase activity in addition to theabove-described genetic modifications.

A reduced activity means both the reduced and the complete eliminationof said activity. Reducing an activity therefore also comprises areduction in the amount of the corresponding protein in the organism upto a complete absence of the corresponding protein, which can be tested,for example, via missing detectability of the corresponding enzymeactivity or missing immunological detectability of the correspondingproteins.

A C24-methyltransferase activity means the enzyme activity of aC24-methyltransferase.

A C24-methyltransferase means a protein which has the enzyme activity ofconverting zymosterol to fecosterol (ergosta-8,24(28)dienol).

Accordingly, C24-methyltransferase activity means the amount ofzymosterol converted or the amount of fecosterol formed by the proteinC24-methyltransferase in a particular time.

In the case of a reduced C24-methyltransferase activity compared to thewild type, thus the amount of zymosterol converted or the amount offecosterol formed by the protein C24-methyltransferase in a particulartime is reduced in comparison with the wild type.

The C24-methyltransferase activity is reduced preferably to at least90%, further preferably to at least 70%, further preferably to at least50%, further preferably to at least 30%, more preferably to at least10%, still more preferably to at least 5%, in particular to 0%, of theC24-methyltransferase activity of the wild type. Therefore, particularpreference is given to eliminating the C24-methyltransferase activity inthe organism.

Δ22-desaturase activity means the enzyme activity of a Δ22-desaturase.

A Δ22-desaturase means a protein which has the enzyme activity ofconverting ergosta-5,7-dienol to ergosta-5,7,22,24-tetraen-313.-ol.

Accordingly, Δ22-desaturase activity means the amount ofergosta-5,7-dienol converted or the amount ofergosta-5,7,22,24-tetraen-313.-ol formed by the protein Δ22-desaturasein a particular time.

In the case of a reduced Δ22-desaturase activity compared to the wildtype, thus the amount of ergosta-5,7-dienol converted or the amount ofergosta-5,7,22,24-tetraen-313.-ol formed by the protein Δ22-desaturasein a particular time is reduced in comparison with the wild type.

The Δ22-desaturase activity is reduced preferably to at least 90%,further preferably to at least 70%, further preferably to at least 50%,further preferably to at least 30%, more preferably to at least 10%,still more preferably to at least 5%, in particular to 0%, of theΔ22-desaturase activity of the wild type. Therefore, particularpreference is given to eliminating the Δ22-desaturase activity in theorganism.

The reduction in C24-methyltransferase activity and/or Δ22-desaturaseactivity may be carried out independently of one another by differentcell-biological mechanisms, for example by inhibiting the correspondingactivity at the protein level, for example by adding inhibitors of thecorresponding enzymes or by reducing gene expression of thecorresponding nucleic acids encoding a C24-methyltransferase orΔ22-desaturase, compared to the wild type.

In a particularly preferred embodiment of the method of the invention,the C24-methyltransferase activity and/or the Δ22-desaturase activityare reduced compared to the wild type by reducing the gene expression ofthe corresponding nucleic acids encoding a C24-methyltransferase orΔ22-desaturase.

Likewise, gene expression of the nucleic acids encoding aC24-methyltransferase or Δ22-desaturase may be reduced compared to thewild type in various ways, for example by

a) introducing nucleic acid sequences which can be transcribed to anantisense nucleic acid sequence which is capable of inhibiting theC24-methyltransferase activity and/or Δ22-desaturase activity, forexample by inhibiting the expression of endogenous C24-methyltransferaseand/or Δ22-desaturase activity,

b) overexpression of homologous C24-methyltransferase nucleic acidsequences and/or Δ22-desaturase nucleic acid sequences, which leads tocosuppression,

c) introducing nonsense mutations into the endogene by means ofintroducing RNA/DNA oligonucleotides into the organism,

d) introducing specific DNA-binding factors, for example factors of thezinc finger transcription factor type, which cause a reduction in geneexpression or

e) generating knockout mutants, for example with the aid of T-DNAmutagenesis or homologous recombination.

In a preferred embodiment of the method of the invention, geneexpression of the nucleic acids encoding a C24-methyltransferase orΔ22-desaturase is reduced by generating knockout mutants, particularlypreferably by homologous recombination.

Therefore, preference is given to using an organism which has nofunctional C24-methyltransferase gene and/or Δ22-desaturase gene.

In a preferred embodiment, knockout mutants are generated, i.e. theC24-methyltransferase-gene target locus and/or the Δ22-desaturase-genetarget locus are deleted with simultaneous integration of an expressioncassette containing at least one of the nucleic acids described above orbelow, which encode a protein whose activity is increased in comparisonwith the wild type, by homologous recombination.

For this purpose, it is possible to use nucleic acid constructs which,in addition to the expression cassettes described below which containpromoter, coding sequence and, where appropriate, terminator and inaddition to a selection marker at the 3′ and 5′ ends, described below,contain nucleic acid sequences which are identical to nucleic acidsequences at the start and the end of the gene to be deleted.

After selection by recombinase systems, the selection marker maypreferably be removed again, for example via loxP signals at the 3′ and5′ ends of the selection marker, using a Cre recombinase (Cre-loxPsystem).

In the preferred organism Saccharomyces cerevisiae, theC24-methyltransferase gene is the gene ERG6 (SEQ. ID. NO. 19). SEQ. ID.NO. 20 represents the corresponding Saccharomyces cerevisiaeC24-methyltransferase (Hardwick, K. G. et al.: SED6 is identical toERG6, and encodes a putative methyltransferase required for ergosterolsynthesis. Yeast. February; 10(2):265-9).

In the preferred organism Saccharomyces cerevisiae, the Δ22-desaturasegene is the gene ERG5 (SEQ. ID. NO. 21). SEQ. ID. NO. 22 represents thecorresponding Saccharomyces cerevisiae Δ22-desaturase (Skaggs, B. A. etal: Cloning and characterization of the Saccharomyces cerevisiae C-22sterol desaturase gene, encoding a second cytochrome P-450 involved inergosterol biosynthesis, Gene. 1996 Feb. 22; 169(1):105-9).

In a further preferred embodiment of the method of the invention,organisms are cultured which have, in addition to the above-describedmodifications, an increased activity of at least one of the activitiesselected from the group consisting of HMG-CoA-reductase activity,lanosterol-C14-demethylase activity, squalene-epoxidase activity,squalene-synthetase activity and sterol-acyltransferase activity,compared to the wild type.

HMG-CoA-reductase activity means the enzyme activity of an HMG-CoAreductase (3-hydroxy-3-methylglutaryl-coenzyme-A reductase).

HMG-CoA reductase means a protein which has the enzyme activity ofconverting 3-hydroxy-3-methylglutaryl-coenzyme A to mevalonate.

Accordingly, HMG-CoA-reductase activity means the amount of3-hydroxy-3-methylglutaryl-coenzyme A converted or the amount ofmevalonate formed by the protein HMG-CoA reductase in a particular time.

In the case of an increased HMG-CoA-reductase activity compared to thewild type, thus the amount of 3-hydroxy-3-methylglutaryl-coenzyme Aconverted or the amount of mevalonate formed by the protein HMG-CoAreductase in a particular time is increased in comparison with the wildtype.

This increase in HMG-CoA-reductase activity is preferably at least 5%,further preferably at least 20%, further preferably at least 50%,further preferably at least 100%, more preferably at least 300%, stillmore preferably at least 500%, in particular at least 600%, of theHMG-CoA-reductase activity of the wild type.

Lanosterol C14-demethylase activity means the enzyme activity of alanosterol C14-demethylase.

Lanosterol C14-demethylase means a protein which has the enzyme activityof converting lanosterol to 4,4-dimethylcholesta-8,14,24-trienol.

Accordingly, lanosterol C14-demethylase activity means the amount oflanosterol converted or the amount of4,4-dimethylcholesta-8,14,24-trienol formed by the protein lanosterolC14-demethylase in a particular time.

In the case of an increased lanosterol C14-demethylase activity comparedto the wild type, thus the amount of lanosterol converted or the amountof 4,4-dimethylcholesta-8,14,24-trienol formed by the protein lanosterolC14-demethylase in a particular time is increased in comparison with thewild type.

This increase in lanosterol C14-demethylase activity is preferably atleast 5%, further preferably at least 20%, further preferably at least50%, further preferably at least 100%, more preferably at least 300%,still more preferably at least 500%, in particular at least 600%, of thelanosterol C14-demethylase activity of the wild type.

Squalene-epoxidase activity means the enzyme activity of a squaleneepoxidase.

Squalene epoxidase means a protein which has the enzyme activity ofconverting squalene to squalene epoxide.

Accordingly, squalene-epoxidase activity means the amount of squaleneconverted or the amount of squalene epoxide formed by the proteinsqualene epoxidase in a particular time.

In the case of an increased squalene-epoxidase activity compared to thewild type, thus the amount of squalene converted or the amount ofsqualene epoxide formed by the protein squalene epoxidase in aparticular time is increased in comparison with the wild type.

This increase in squalene-epoxidase activity is preferably at least 5%,further preferably at least 20%, further preferably at least 50%,further preferably at least 100%, more preferably at least 300%, stillmore preferably at least 500%, in particular at least 600%, of thesqualene-epoxidase activity of the wild type.

Squalene-synthetase activity means the enzyme activity of a squalenesynthetase.

Squalene synthetase means a protein which has the enzyme activity ofconverting farnesyl-pyrophosphate to squalene.

Accordingly, squalene-synthetase activity means the amount offarnesyl-pyrophosphate converted or the amount of squalene formed by theprotein squalene synthetase in a particular time.

In the case of an increased squalene-synthetase activity compared to thewild type, thus the amount of farnesyl-pyrophosphate converted or theamount of squalene formed by the protein squalene synthetase in aparticular time is increased in comparison with the wild type.

This increase in squalene-synthetase activity is preferably at least 5%,further preferably at least 20%, further preferably at least 50%,further preferably at least 100%, more preferably at least 300%, stillmore preferably at least 500%, in particular at least 600%, of thesqualene-synthetase activity of the wild type.

Sterol-acyltransferase activity means the enzyme activity of a sterolacyltransferase.

Sterol acyltransferase means a protein which has the enzyme activity ofconverting 7-dehydrocholesterol to corresponding acetylated7-dehydrocholesterol.

Accordingly, sterol-acyltransferase activity means the amount of7-dehydrocholesterol converted or the amount of acetylated7-dehydrocholesterol formed by the protein sterol acyltransferase in aparticular time.

In the case of an increased sterol-acyltransferase activity compared tothe wild type, thus the amount of 7-dehydrocholesterol converted or theamount of acetylated 7-dehydrocholesterol formed by the protein sterolacyltransferase in a particular time is increased in comparison with thewild type.

This increase in sterol-acyltransferase activity is preferably at least5%, further preferably at least 20%, further preferably at least 50%,further preferably at least 100%, more preferably at least 300%, stillmore preferably at least 500%, in particular at least 600%, of thesterol-acyltransferase activity of the wild type.

In a preferred embodiment, the HMG-CoA-reductase activity is increasedcompared to the wild type by increasing the gene expression of a nucleicacid encoding an HMG-CoA reductase.

In a particularly preferred embodiment of the method of the invention,gene expression of a nucleic acid encoding an HMG-CoA reductase isincreased by introducing into the organism a nucleic acid constructcomprising an HMG-CoA reductase-encoding nucleic acid whose expressionin said organism is subject to a reduced regulation, in comparison withthe wild type.

A reduced regulation in comparison with the wild type means a reducedregulation and, preferably, no regulation at the expression or proteinlevel, in comparison with the above-defined wild type.

The reduced regulation may be achieved preferably by a promoter which isfunctionally linked with the coding sequence in the nucleic acidconstruct and which is subject to a reduced regulation in the organism,in comparison with the wild-type promoter.

For example, the medium ADH promoter in yeast is subject only to areduced regulation and is therefore particularly preferred as promoterin the above-described nucleic acid construct.

This promoter fragment of the ADH12s promoter, also referred to as ADH1hereinbelow, exhibits nearly constitutive expression (Ruohonen L,Penttila M, Keranen S. (1991) Optimization of Bacillus α-amylaseproduction by Saccharomyces cerevisiae. Yeast. May-June; 7(4):337-462;Lang C, Looman A C. (1995) Efficient expression and secretion ofAspergillus niger RH5344 polygalacturonase in Saccharomyces cerevisiae.Appl Microbiol Biotechnol. December; 44(1-2):147-56) so thattranscriptional regulation no longer proceeds via intermediates ofergosterol biosynthesis.

Other preferred promoters with reduced regulation are constitutivepromoters such as, for example, the yeast TEF1 promoter, the yeast GPDpromoter or the yeast PGK promoter (Mumberg D, Muller R, Funk M. (1995)Yeast vectors for the controlled expression of heterologous proteins indifferent genetic backgrounds. Gene. 1995 Apr. 14; 156(1):119-22; Chen CY, Oppermann H, Hitzeman R A. (1984) Homologous versus heterologous geneexpression in the yeast, Saccharomyces cerevisiae. Nucleic Acids Res.December 11; 12(23):8951-70).

In a further preferred embodiment, reduced regulation can be achieved byusing as an HMG-CoA reductase-encoding nucleic acid a nucleic acid whoseexpression in the organism is subject to a reduced regulation, incomparison with the orthologous nucleic acid intrinsic to said organism.

Particular preference is given to using as an HMG-CoA reductase-encodingnucleic acid a nucleic acid which encodes only the catalytic region ofHMG-CoA reductase (truncated (t-) HMG-CoA reductase). This nucleic acid(t-HMG), described in EP 486 290 and WO 99/16886 encodes only thecatalytically active part of HMG-CoA reductase, with the membrane domainresponsible for regulation at the protein level missing. This nucleicacid is thus subject to a reduced regulation, in particular in yeast,and leads to an increase in gene expression of HMG-CoA reductase.

In a particularly preferred embodiment, nucleic acids are introduced,preferably via the above-described nucleic acid construct, which encodeproteins comprising the amino acid sequence SEQ. ID. NO. 24 or asequence derived from this sequence by substitution, insertion ordeletion of amino acids, which is at least 30% identical at the aminoacid level to the sequence SEQ ID. NO. 24, and having the enzymeproperty of an HMG-CoA reductase.

The sequence SEQ ID NO. 24 is the amino acid sequence of the truncatedHMG-CoA reductase (t-HMG).

Further examples of HMG-CoA reductases and thus also of the t-HMG-CoAreductases reduced to the catalytic region or of the coding genes canreadily be found, for example, for various organisms whose genomicsequence is known by comparing the homology of the amino acid sequencesor of the corresponding backtranslated nucleic acid sequences fromdatabases with the sequence SEQ ID. No. 24.

Further examples of HMG-CoA reductases and thus also of the t-HMG-CoAreductases reduced to the catalytic region and of the coding genes canfurthermore readily be found for various organisms whose genomicsequence is unknown by hybridization techniques and PCR techniques in amanner known per se, for example starting from the sequence SEQ ID NO.23.

Particular preference is given to using as a truncated HMG-CoAreductase-encoding nucleic acid a nucleic acid comprising the sequenceSEQ ID NO. 23.

In a particularly preferred embodiment, the reduced regulation isachieved by using as an HMG-CoA reductase-encoding nucleic acid anucleic acid whose expression in the organism is subject to a reducedregulation, in comparison with the orthologous nucleic acid intrinsic tosaid organism, and by using a promoter which is subject to a reducedregulation in said organism, in comparison with the wild-type promoter.

In a preferred embodiment, the lanosterol C14-demethylase activity isincreased compared to the wild type by increasing the gene expression ofa nucleic acid encoding a lanosterol C14-demethylase.

In a further preferred embodiment, gene expression of a nucleic acidencoding a lanosterol C14-demethylase is increased by introducing intothe organism one or more nucleic acids encoding a lanosterolC14-demthylase.

For this purpose, it is possible to use in principle any lanosterolC14-demethylase gene (ERG11), i.e. any nucleic acids encoding alanosterol C14-demethylase. In the case of genomic lanosterolC14-demethylase nucleic acid sequences from eukaryotic sources, whichcontain introns, already processed nucleic acid sequences such as thecorresponding cDNAs are to be used preferably, if the host organism isunable to or cannot be enabled to express the appropriate lanosterolC14-demethylase.

Examples of lanosterol C14-demethylase genes are nucleic acids encodinga lanosterol C14-demethylase of Saccharomyces cerevisiae (Kalb V F,Loper J C, Dey C R, Woods C W, Sutter T R (1986) Isolation of acytochrome P-450 structural gene from Saccharomyces cerevisiae. Gene45(3):237-45), Candida albicans (Lamb D C, Kelly D E, Baldwin B C, GozzoF, Boscott P, Richards W G, Kelly S L (1997) Differential inhibition ofCandida albicans CYP51 with azole antifungal stereoisomers. FEMSMicrobiol Lett 149(1):25-30), Homo sapiens (Stromstedt M, Rozman D,Waterman M R. (1996) The ubiquitously expressed human CYP51 encodeslanosterol 14α-demethylase, a cytochrome P450 whose expression isregulated by oxysterols. Arch Biochem Biophys 1996 May 1; 329(1):73-81c)or Rattus norvegicus, Aoyama Y, Funae Y, Noshiro M, Horiuchi T, YoshidaY. (1994) Occurrence of a P450 showing high homology to yeast lanosterol14-demethylase (P450(14DM)) in the rat liver. Biochem Biophys ResCommun. June 30; 201(3):1320-6).

In this preferred embodiment, thus at least one further lanosterolC14-demethylase gene is present in the transgenic organisms of theinvention, compared to the wild type.

The number of C14-demethylase genes in the transgenic organisms of theinvention is at least two, preferably more than two, particularlypreferably more than three and very particularly preferably more thanfive.

Preference is given to using in the above-described method nucleic acidswhich encode proteins comprising the amino acid sequence SEQ. ID. NO. 26or a sequence derived from this sequence by substitution, insertion ordeletion of amino acids, which is at least 30%, preferably at least 50%,more preferably at least 70%, still more preferably at least 90%, mostpreferably at least 95%, identical at the amino acid level with thesequence SEQ. ID. NO. 26, and having the enzyme property of a lanosterolC14-demethylase.

The sequence SEQ. ID. NO. 26 represents the amino acid sequence ofSaccharomyces cerevisiae lanosterol C14-demethylase.

Further examples of lanosterol C14-demethylases and lanosterolC14-demethylase genes can readily be found, for example, for variousorganisms whose genomic sequence is known by comparing the homology ofthe amino acid sequences or the corresponding backtranslated nucleicacid sequences from databases with the SeQ ID. NO. 26.

Further examples of lanosterol C14-demethylases and lanosterolC14-demethylase genes can furthermore readily be found for variousorganisms whose genomic sequence is unknown, for example starting fromthe sequence SEQ. ID. No. 25, by hybridization techniques and PCRtechniques in a manner known per se.

Accordingly, a protein which is at least 30% identical at the amino acidlevel with the sequence SEQ. ID. NO. 26 means a protein which is atleast 30% identical when comparing its sequence with the sequence SEQ.ID. NO. 26, in particular according to the above program algorithm withthe above set of parameters.

In another preferred embodiment, nucleic acids are introduced intoorganisms, which encode proteins comprising the amino acid sequence ofSaccharomyces cerevisiae lanosterol C14-demethylase (SEQ. ID. NO. 26).

Suitable nucleic acid sequences can be obtained, for example, bybacktranslating the polypeptide sequence according to the genetic code.

Preference is given to using for this those codons which are frequentlyused according to the organism-specific codon usage. Said codon usagecan readily be determined on the basis of computer analyses of otherknown genes of the organisms in question.

If the protein is to be expressed in yeast, for example, it is oftenadvantageous to use the codon usage of yeast for the backtranslation.

In a particularly preferred embodiment, a nucleic acid comprising thesequence SEQ. ID. NO. 25 is introduced into the organism.

The sequence SEQ. ID. NO. 25 represents the genomic DNA of Saccharomycescerevisiae (ORF S0001049), which encodes the lanosterol C14-demethylaseof the sequence SEQ ID NO. 26.

Furthermore, all of the lanosterol C14-demethylase genes mentioned abovecan be prepared in a manner known per se by chemical synthesis from thenucleotide building blocks, for example by fragment condensation ofindividual overlapping complementary nucleic acid building blocks of thedouble helix. The chemical synthesis of oligonucleotides may be carriedout, for example, in a known manner according to the phosphoramiditemethod (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).Annealing of synthetic oligonucleotides and filling-in of gaps with theaid of the Klenow fragment of DNA polymerase and the ligation reactionsand also general cloning methods are described in Sambrook et al.(1989), Molecular cloning: A laboratory manual, Cold Spring HarborLaboratory Press.

In a preferred embodiment, the squalene-epoxidase activity is increasedcompared to the wild type by increasing the gene expression of a nucleicacid encoding a squalene epoxidase.

In a further preferred embodiment, gene expression of a nucleic acidencoding a squalene epoxidase is increased by introducing into theorganism one or more nucleic acids encoding a squalene epoxidase.

For this purpose, it is possible to use in principle anysqualene-epoxidase gene (ERG1), i.e. any nucleic acids encoding asqualene epoxidase. In the case of genomic squalene epoxidase nucleicacid sequences from eukaryotic sources, which contain introns, alreadyprocessed nucleic acid sequences such as the corresponding cDNAs are tobe used preferably, if the host organism is unable to or cannot beenabled to express the appropriate squalene epoxidase.

Examples of nucleic acids encoding a squalene epoxidase are nucleicacids encoding a squalene epoxidase of Saccharomyces cerevisiae(Jandrositz, A., et al (1991) The gene encoding squalene epoxidase fromSaccharomyces cerevisiae: cloning and characterization. Gene107:155-160, of Mus musculus (Kosuga K, Hata S, Osumi T, Sakakibara J,Ono T. (1995) Nucleotide sequence of a cDNA for mouse squaleneepoxidase, Biochim Biophys Acta, February 21; 1260(3):345-8b), of Rattusnorvegicus (Sakakibara J, Watanabe R, Kanai Y, Ono T. (1995) Molecularcloning and expression of rat squalene epoxidase. J Biol Chem January 6;270(1):17-20c) or of Homo sapiens (Nakamura Y, Sakakibara J, Izumi T,Shibata A, Ono T. (1996) Transcriptional regulation of squaleneepoxidase by sterols and inhibitors in HeLa cells., J. Biol. Chem. 1996,Apr. 5; 271(14):8053-6).

In this preferred embodiment, thus at least one further squaleneepoxidase is present in the transgenic organisms of the invention,compared to the wild type.

The number of squalene-epoxidase genes in the transgenic organisms ofthe invention is at least two, preferably more than two, particularlypreferably more than three and very particularly preferably more thanfive.

Preference is given to using in the above-described method nucleic acidswhich encode proteins comprising the amino acid sequence SEQ. ID. NO. 28or a sequence derived from this sequence by substitution, insertion ordeletion of amino acids, which is at least 30%, preferably at least 50%,more preferably at least 70%, still more preferably at least 90%, mostpreferably at least 95%, identical at the amino acid level with thesequence SEQ. ID. NO. 28, and having the enzyme property of a squaleneepoxidase.

The sequence SEQ. ID. NO. 28 represents the amino acid sequence ofSaccharomyces cerevisiae squalene epoxidase.

Further examples of squalene epoxidases and squalene-epoxidase genes canreadily be found, for example, for various organisms whose genomicsequence is known by comparing the homology of the amino acid sequencesor the corresponding backtranslated nucleic acid sequences fromdatabases with the SeQ ID. NO. 28.

Further examples of squalene epoxidases and squalene-epoxidase genes canfurthermore readily be found for various organisms whose genomicsequence is unknown, for example starting from the sequence SEQ. ID. No.27, by hybridization techniques and PCR techniques in a manner known perse.

In another preferred embodiment, nucleic acids are introduced intoorganisms, which encode proteins comprising the amino acid sequence ofSaccharomyces cerevisiae squalene epoxidase (SEQ. ID. NO. 28).

Suitable nucleic acid sequences can be obtained, for example, bybacktranslating the polypeptide sequence according to the genetic code.

Preference is given to using for this those codons which are frequentlyused according to the organism-specific codon usage. Said codon usagecan readily be determined on the basis of computer analyses of otherknown genes of the organisms in question.

If the protein is to be expressed in yeast, for example, it is oftenadvantageous to use the codon usage of yeast for backtranslation.

In a particularly preferred embodiment, a nucleic acid comprising thesequence SEQ. ID. NO. 27 is introduced into the organism.

The sequence SEQ. ID. NO. 27 represents the genomic DNA of Saccharomycescerevisiae (ORF YGR175C), which encodes the squalene epoxidase of thesequence SEQ ID NO. 28.

Furthermore, all of the squalene-epoxidase genes mentioned above can beprepared in a manner known per se by chemical synthesis from thenucleotide building blocks, for example by fragment condensation ofindividual overlapping complementary nucleic acid building blocks of thedouble helix. The chemical synthesis of oligonucleotides may be carriedout, for example, in a known manner according to the phosphoramiditemethod (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).Annealing of synthetic oligonucleotides and filling-in of gaps with theaid of the Klenow fragment of DNA polymerase and the ligation reactionsand also general cloning methods are described in Sambrook et al.(1989), Molecular cloning: A laboratory manual, Cold Spring HarborLaboratory Press.

In a preferred embodiment, the squalene-synthetase activity is increasedcompared to the wild type by increasing the gene expression of a nucleicacid encoding a squalene synthetase.

In a further preferred embodiment, gene expression of a nucleic acidencoding a squalene synthetase is increased by introducing into theorganism one or more nucleic acids encoding a squalene synthetase.

For this purpose, it is possible to use in principle anysqualene-synthetase gene (ERG9), i.e. any nucleic acids encoding asqualene synthetase. In the case of genomic squalene synthetase nucleicacid sequences from eukaryotic sources, which contain introns, alreadyprocessed nucleic acid sequences such as the corresponding cDNAs are tobe used preferably, if the host organism is unable to or cannot beenabled to express the appropriate squalene synthetase.

Examples of nucleic acids encoding a squalene synthetase are nucleicacids encoding a Saccharomyces cerevisiae squalene synthetase (ERG9)(Jennings, S. M., (1991): Molecular cloning and characterization of theyeast gene for squalene synthetase. Proc Natl Acad Sci USA. July 15;88(14):6038-42), nucleic acids encoding a Botryococcus braunii Okadasqualene synthetase (Devarenne, T. P. et al.: Molecular characterizationof squalene synthetase from the green microalga Botryococcus braunii,raceB, Arch. Biochem. Biophys. 2000, Jan. 15, 373(2):307-17), nucleicacids encoding a Potato tuber squalene synthetase (Yoshioka H. et al.:cDNA cloning of sesquiter penecyclase and squalene synthase, andexpression of the genes in potato tuber infected with Phytophthorainfestans, Plant. Cell. Physiol. 1999, September; 40(9):993-8) andnucleic acids encoding a Glycyrrhiza glabra squalene synthetase(Hayashi, H. et al.: Molecular cloning and characterization of two cDNAsfor Glycyrrhiza glabra squalene synthase, Biol. Pharm. Bull. 1999,September; 22(9):947-50).

In this preferred embodiment, thus at least one furthersqualene-synthetase gene is present in the transgenic organisms of theinvention, compared to the wild type.

The number of squalene-synthetase genes in the transgenic organisms ofthe invention is at least two, preferably more than two, particularlypreferably more than three and very particularly preferably more thanfive.

Preference is given to using in the above-described method nucleic acidswhich encode proteins comprising the amino acid sequence SEQ. ID. NO. 30or a sequence derived from this sequence by substitution, insertion ordeletion of amino acids, which is at least 30%, preferably at least 50%,more preferably at least 70%, still more preferably at least 90%, mostpreferably at least 95%, identical at the amino acid level with thesequence SEQ. ID. NO. 30, and having the enzyme property of a squalenesynthetase.

The sequence SEQ. ID. NO. 30 represents the amino acid sequence ofSaccharomyces cerevisiae squalene synthetase (ERG9).

Further examples of squalene synthetases and squalene-synthetase genescan readily be found, for example, for various organisms hose genomicsequence is known by comparing the homology of the amino acid sequencesor the corresponding backtranslated nucleic acid sequences fromdatabases with the SeQ ID. NO. 30.

Further examples of squalene synthetases and squalene-synthetase genescan furthermore readily be found for various organisms whose genomicsequence is unknown, for example starting from the sequence SEQ. ID. No.29, by hybridization techniques and PCR techniques in a manner known perse.

In another preferred embodiment, nucleic acids are introduced intoorganisms, which encode proteins comprising the amino acid sequence ofSaccharomyces cerevisiae squalene synthetase (SEQ. ID. NO. 30).

Suitable nucleic acid sequences can be obtained, for example, bybacktranslating the polypeptide sequence according to the genetic code.

Preference is given to using for this those codons which are frequentlyused according to the organism-specific codon usage. Said codon usagecan readily be determined on the basis of computer analyses of otherknown genes of the organisms in question.

If the protein is to be expressed in yeast, for example, it is oftenadvantageous to use the codon usage of yeast for the backtranslation.

In a particularly preferred embodiment, a nucleic acid comprising thesequence SEQ. ID. NO. 29 is introduced into the organism.

The sequence SEQ. ID. NO. 29 represents the genomic DNA of Saccharomycescerevisiae (ORF YHR190W), which encodes the squalene synthetase of thesequence SEQ ID NO. 30.

Furthermore, all of the squalene-synthetase genes mentioned above can beprepared in a manner known per se by chemical synthesis from thenucleotide building blocks, for example by fragment condensation ofindividual overlapping complementary nucleic acid building blocks of thedouble helix. The chemical synthesis of oligonucleotides may be carriedout, for example, in a known manner according to the phosphoramiditemethod (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).Annealing of synthetic oligonucleotides and filling-in of gaps with theaid of the Klenow fragment of DNA polymerase and the ligation reactionsand also general cloning methods are described in Sambrook et al.(1989), Molecular cloning: A laboratory manual, Cold Spring HarborLaboratory Press.

In a preferred embodiment, the sterol-acyltransferase activity isincreased compared to the wild type by increasing the gene expression ofa nucleic acid encoding a sterol acyltransferase.

In a further preferred embodiment, gene expression of a nucleic acidencoding a sterol acyltransferase is increased by introducing into theorganism one or more nucleic acids encoding a sterol acyltransferase.

For this purpose, it is possible to use in principle anysterol-acyltransferase gene (SAT1 or SAT2), i.e. any nucleic acidsencoding a sterol acyltransferase.

In the case of genomic sterol acyltransferase nucleic acid sequencesfrom eukaryotic sources, which contain introns, already processednucleic acid sequences such as the corresponding cDNAs are to be usedpreferably, if the host organism is unable to or cannot be enabled toexpress the appropriate sterol acyltransferase.

Examples of nucleic acids encoding a sterol acyltransferase are nucleicacids encoding a Saccharomyces cerevisiae sterol acyltransferase (SAT1)or (SAT2) (Yang, H.: Sterol esterification in yeast: a two-gene process.Science. 1996 May 31; 272(5266):1353-6), a further nucleic acid encodinga further Saccharomyces cerevisiae sterol acyltransferase (J. Biol.Chem. 1996, Sep. 27; 271(39):24157-63), nucleic acids encoding a humansterol acyltransferase (Chang, C. C. et al., Molecular cloning andfunctional expression of human acyl-coenzyme A:cholesterolacyltransferase cDNA in mutant Chinese hamster ovary cells, J. Biol.Chem. 1993, Oct. 5; 268(28):20747-55) and nucleic acids encoding amurine sterol acyltransferase (Uelmen, P. J.: Tissue-specific expressionand cholesterol regulation of acylcoenzyme A:cholesterol acyltransferase(ACAT) in mice. Molecular cloning of mouse ACAT cDNA, chromosomallocalization, and regulation of ACAT in vivo and in vitro, J. Biol.Chem. 1995 Nov. 3; 270(44):26192-201).

In this preferred embodiment, thus at least one furthersterol-acyltransferase gene is present in the transgenic organisms ofthe invention, compared to the wild type.

The number of sterol-acyltransferase genes in the transgenic organismsof the invention is at least two, preferably more than two, particularlypreferably more than three and very particularly preferably more thanfive.

Preference is given to using in the above-described method nucleic acidswhich encode proteins comprising the amino acid sequence SEQ. ID. NO. 32or SEQ ID NO. 50 or a sequence derived from these sequences bysubstitution, insertion or deletion of amino acids, which is at least30%, preferably at least 50%, more preferably at least 70%, still morepreferably at least 90%, most preferably at least 95%, identical at theamino acid level with the sequence SEQ. ID. NO. 32 or SEQ. ID. NO. 50,and having the enzyme property of a sterol acyltransferase.

The sequence SEQ. ID. NO. 32 represents the amino acid sequence ofSaccharomyces cerevisiae sterol acyltransferase SAT1.

The sequence SEQ. ID. NO. 50 represents the amino acid sequenceSaccharomyces cerevisiae sterol acyltransferase SAT2.

SAT 1 and SAT2 differ from one another by a different substratespecificity.

Further examples of sterol acyltransferases and sterol-acyltransferasegenes can readily be found, for example, for various organisms whosegenomic sequence is known by comparing the homology of the amino acidsequences or the corresponding backtranslated nucleic acid sequencesfrom databases with the SeQ ID. NO. 32 or 50.

Further examples of sterol acyltransferase and sterol-acyltransferasegenes can furthermore readily be found for various organisms whosegenomic sequence is unknown, for example starting from the sequence SEQ.ID. No. 31 or 49, by hybridization techniques and PCR techniques in amanner known per se.

In another preferred embodiment, nucleic acids are introduced intoorganisms, which encode proteins comprising the amino acid sequence ofSaccharomyces cerevisiae sterol acyltransferase SAT1 (SEQ. ID. NO. 32)or Saccharomyces cerevisiae sterol acyltransferase SAT2 (SEQ. ID. NO.50).

Suitable nucleic acid sequences can be obtained, for example, bybacktranslating the polypeptide sequence according to the genetic code.

Preference is given to using for this those codons which are frequentlyused according to the organism-specific codon usage. Said codon usagecan readily be determined on the basis of computer analyses of otherknown genes of the organisms in question.

If the protein is to be expressed in yeast, for example, it is oftenadvantageous to use the codon usage of yeast for the backtranslation.

In a particularly preferred embodiment, a nucleic acid comprising thesequence SEQ. ID. NO. 31 or 49 is introduced into the organism.

The sequence SEQ. ID. NO. 31 represents the genomic DNA of Saccharomycescerevisiae (ORF YNR019W), which encodes the sterol acyltransferase SAT1of the sequence SEQ ID NO. 32.

The sequence SEQ. ID. NO. 49 represents the genomic DNA of Saccharomycescerevisiae (ORF YCR048W), which encodes the sterol acyltransferase SAT2of the sequence SEQ ID NO. 50.

Furthermore, all of the sterol-acyltransferase genes mentioned above canbe prepared in a manner known per se by chemical synthesis from thenucleotide building blocks, for example by fragment condensation ofindividual overlapping complementary nucleic acid building blocks of thedouble helix. The chemical synthesis of oligonucleotides may be carriedout, for example, in a known manner according to the phosphoramiditemethod (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).Annealing of synthetic oligonucleotides and filling-in of gaps with theaid of the Klenow fragment of DNA polymerase and the ligation reactionsand also general cloning methods are described in Sambrook et al.(1989), Molecular cloning: A laboratory manual, Cold Spring HarborLaboratory Press.

According to the invention, organisms mean, for example, bacteria, inparticular bacteria of the genus Bacillus, Escherichia coli,Lactobacillus spec. or Streptomyces spec., for example yeasts, inparticular yeasts of the genus Saccharomyces cerecisiae, Pichia pastorisor Klyveromyces spec.

for example fungi, in particular fungi of the genus Aspergillus spec.,Penicillium spec. or Dictyostelium spec.

and also, for example, insect cell lines, which are capable, either aswild type or owing to previous genetic modification, of producingzymosterol and/or the biosynthetic intermediates and/or secondaryproducts thereof.

Particularly preferred organisms are yeasts, in particular those of thespecies Saccharomyces cerevisiae, in particular the yeast strainsSaccharomyces cerevisiae AH22, Saccharomyces cerevisiae GRF,Saccharomyces cerevisiae DBY747 and Saccharomyces cerevisiae BY4741.

In the case of yeasts as organisms or genetically modified organisms, itis possible, as mentioned above, to increase at least one of theactivities selected from the group consisting of Δ8-Δ7-isomeraseactivity, Δ5-desaturase activity and Δ24-reductase activity byoverexpressing the corresponding nucleic acids.

The overexpression may be carried out both homologously by introducingnucleic acids intrinsic to yeast and heterologously by introducingnucleic acids from other organisms, in particular mammals, or natural orartificial variants derived therefrom into the yeast. Preference isgiven to using mammalian genes in yeasts, since these genes have abetter substrate specificity with respect to 7-dehydrocholesterol.

The Δ8-Δ7-isomerase activity, Δ5-desaturase activity, Δ24-reductaseactivity, C24-methyltransferase activity, Δ22-desaturase activity,HMG-CoA-reductase activity, lanosterol-C14-demethylase activity,squalene-epoxidase activity, squalene-synthetase activity andsterol-acyltransferase activity of the genetically modified organism ofthe invention and of the reference organism is determined under thefollowing conditions:

The activity of HMG-CoA reductase is determined as described in Th.Polakowski, Molekularbiologische Beeinflussung desErgosterolstoffwechsels der Hefe Saccharomyces cerevisiae [influencingthe ergosterol metabolism of the yeast Saccharomyces cerevisiae bymolecular biological means], Shaker-Verlag, Aachen 1999, ISBN3-8265-6211-9, beschrieben.

According to this, 10⁹ yeast cells of a 48 h culture are harvested bycentrifugation (3500.times.g, 5 min) and washed in 2 ml of buffer I (100mM potassium phosphate buffer, pH 7.0). The cell pellet is taken up in500 μl of buffer 1 (cytosolic proteins) or 2 (100 mM potassium phosphatebuffer pH 7.0; 1% Triton X-100) (total proteins), and 1 μl of 500 mMPMSF in isopropanol is added. 500 μl of glass beads (d=0.5 mm) are addedto the cells and the cells are disrupted by vortexing 5.times. for oneminute each. The liquid between the glass beads is transferred to a newEppendorf vessel. Cell debris and membrane components are removed bycentrifugation (14000.times.g; 15 min). The supernatant is transferredto a new Eppendorf vessel and represents the protein fraction.

The activity of HMG-CoA reductase is determined by measuring NADPH+H⁺consumption during the reduction of 3-hydroxy-3-methylglutaryl-CoA whichis added as substrate.

In a 1000 μl assay mixture, 20 μl of yeast protein isolate are combinedwith 910 μl of buffer I; 50 μl of 0.1 M DTT and 10 μl of 16 mM NADPH+H⁺.The mixture is adjusted to 30.degree. C. and measured in aspectrophotometer at 340 nm for 7.5 min. The decrease in NADPH, which ismeasured over this period, is the rate of degradation without additionof substrate and is taken into account as background.

Subsequently, substrate (10 μl of 30 mM HMG-CoA) is added, andmeasurement continues for another 7.5 min. The HMG-CoA-reductaseactivity is calculated by determining the specific rate of NADPHdegradation.

The activity of lanosterol C14-demethylase is determined as described inOmura, T and Sato, R. (1964) The carbon monoxide binding pigment inliver microsomes. J. Biol. Chem. 239, 2370-2378. In this assay, theamount of P450 enzyme as holoenzyme with bound heme can besemi-quantified. The (active) holoenzyme (with heme) can be reduced byCO and only the CO-reduced enzyme has an absorption maximum at 450 nm.Thus the absorption maximum at 450 nm is a measure for lanosterolC14-demethylase activity.

The activity is determined by diluting a microsomal fraction (4-10 mg/mlprotein in 100 mM potassium phosphate buffer) 1:4 so that the proteinconcentration used in the assay is 2 mg/ml. The assay is carried outdirectly in a cuvette.

A spatula tipful of dithionite (S₂O₄Na₂) is added to the microsomes. Thebaseline is recorded in the 380-500 nm region in a spectrophotometer.

Subsequently, approx. 20-30 CO bubbles are passed through the sample.The absorption is then measured in the same region. The absorption levelat 450 nm corresponds to the amount of P450 enzyme in the assay mixture.

The activity of squalene epoxidase is determined as described in LeberR, Landl K, Zinser E, Ahorn H, Spok A, Kohlwein S D, Turnowsky F, DaumG. (1998) Dual localization of squalene epoxidase, Erg1p, in yeastreflects a relationship between the endoplasmic reticulum and lipidparticles, Mol. Biol. Cell. 1998, February; 9(2):375-86.

In this method, a total volume of 500 μl contains from 0.35 to 0.7 mg ofmicrosomal protein or from 3.5 to 75 μg of lipid-particle protein in 100mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1 mM FAD, 3 mM NADPH, 0.1 mM squalene2,3-epoxidase cyclase inhibitor U18666A, 32 μM [^(3H)]squalene dispersedin 0.005% Tween 80.

The assay is carried out at 30.degree. C. After 10 minutes ofpretreatment, the reaction is started by adding squalene and stoppedafter 15, 30 or 45 min by lipid extraction with 3 ml ofchloroform/methanol (2:1 vol/vol) and 750 μl of 0.035% MgCl₂.

The lipids are dried under nitrogen and redissolved in 0.5 ml ofchloroform/methanol (2:1 vol/vol). For thin layer chromatography,portions are applied to a Silica Gel 60 μlate (0.2 mm) and fractionatedusing chloroform as eluent. The positions containing[^(3H)]2,3-oxidosqualene and [^(3H)]squalene were scraped off andquantified in a scintillation counter.

The Δ8-Δ7-isomerase activity is determined, with a slight modification,as described in Silve S. et al.: Emopamil-binding Protein, a MammalianProtein That Binds a Series of Structurally Diverse NeuroprotectiveAgents, Exhibits 8-7 Sterol Isomerase Activity in Yeast. J Biol Chem1996 Sep. 13; 271(37):22434-40:

Microsomes prepared from a culture volume of 10 ml are incubated in thepresence of 75 μM cholesta-8-en-3-ol at 30.degree. C. for 3 h. Thesterols are then extracted with 4 times 5 ml of hexane and purified.Aliquots are analyzed by means of GC/MS.

The Δ5-desaturase activity is determined, with slight modification, asdescribed in Nishi, S. et al. (2000): cDNA cloning of the mammaliansterol C5-desaturase and the expression in yeast mutant. Biochim.Biophys. Acta 1490(1-2), 106-108:

Microsomes prepared from a culture volume of 10 ml are incubated in thepresence of 75 μM lathosterol and 2 mM NADH at 30.degree. C. for 3 h.The sterols are then extracted with 4 times 5 ml of hexane and purified.Aliquots are analyzed by means of GC/MS.

The Δ24-reductase activity can be determined as described below:

Microsomes prepared from a culture volume of 10 ml are incubated in thepresence of 75 μM cholesta-5,7,24-trienol at 30.degree. C. for 3 h. Thesterols are then extracted with 4 times 5 ml of hexane and purified.Aliquots are analyzed by means of GC/MS.

The C24-methyltransferase activity can be determined as described below:

80% of the protein Erg6p (C24-methyltransferase) are detectable in lipidparticles in the yeast (Athenstaedt K, Zweytick D, Jandrositz A,Kohlwein S D, Daum G: Identification and characterization of major lipidparticle proteins of the yeast Saccharomyces cerevisiae. J. Bacteriol.1999 October; 181(20):6441-8). The enzyme activity is determined bypreparing lipid particles from a culture volume (48 h) of 100 ml(according to a method described in Athenstaedt K, Zweytick D,Jandrositz A, Kohlwein S D, Daum G: Identification and characterizationof major lipid particle proteins of the yeast Saccharomyces cerevisiae.J. Bacteriol. 1999 October; 181(20):6441-8).

The protein content is determined by a Biorad enzyme assay and 3 mg ofprotein are used in a volume of 500 μl for each assay mixture. 50 μM[methyl-³H₃]—S-adenosylmethionine and 50 μM zymosterol are added to theassay mixture which is then incubated at 35.degree. C. for 10 min.Subsequently, the same volume (500 μl) of chloroform/methanol (4:1) isadded and the sterols are then extracted.

The proportion of zymosterol with incorporated[methyl-³H₃]—S-adenosylmethionine can be determined by means ofscintillation measurement, since chloroform/methanol extraction extractsonly lipid-soluble substances. For quantification, the radioactivedecays are likewise determined for 50 μM[methyl-³H₃]—S-adenosylmethionine by means of scintillation measurement.

This method is a modification of the method described in Nes W D, Guo D,Zhou W.: Substrate-based inhibitors of the(S)-adenosyl-L-methionine:Δ24(25)- to Δ24(28)-sterol methyl transferasefrom Saccharomyces cerevisiae, Arch. Biochem. Biophys. 1997 Jun. 1;342(1):68-81.

The activity of Δ22-desaturase (ERG5p) can be determined as describedbelow:

Various concentrations of Ergosta-5,7-dienol, purified from S.cerevisiae erg5 mutants (Parks et al, 1985. Yeast sterols.yeast mutantsas tools for the study of sterol metabolism. Methods Enzymol.111:333-346) and 50 μg of dilauroylphosphatidylcholine are mixed andtreated with ultrasound until a white suspension is formed. Preparedmicrosomes are added (1 ml)(3 mg/ml protein). NADPH (1 mM finalconcentration) is added to the assay mixture to start the enzymereaction. The mixture is incubated at 37.degree. C. for 20 min. Thereaction is stopped by adding 3 ml of methanol and sterols arehydrolyzed by adding 2 ml of 60% (wt/vol) KOH in water. The mixture isincubated at 90.degree. C. for 2 h. After cooling, the mixture isextracted three times with 5 ml of hexane and concentrated in a rotaryevaporator. Subsequently, the sterols are silylated withbis(trimethylsilyl)trifluoroacetamide (50 μl in 50 μl toluene) at60.degree. C. for 1 h. The sterols are analyzed by gaschromatography-mass spectrometry (GC-MS) (for example Model VG 12-250gas chromatograph-mass spectrometer; VG Biotech, Manchester, UnitedKingdom). The resultant Δ22-desaturated intermediate can be identifieddepending on the amount of substrate used. Microsomes which are notincubated with substrate serve as reference.

This method is a modification of the method described in Lamb et al:Purification, reconstitution, and inhibition of cytochrome P-450 sterolΔ22-desaturase from the pathogenic fungus Candida glabrata. AntimicrobAgents Chemother. 1999 July; 43(7):1725-8.

The squalene-synthetase activity can be determined as described below:

The assays contain 50 mM MOPS, pH 7.2, 10 mM MgCl₂, 1% (v/v) Tween-80,10% (v/v) 2-propanol, 1 mM DTT, 1 mg/mL BSA, NADPH, FPP (or PSPP) andmicrosomes (protein content 3 mg) in a total volume of 200 μl in glasstubes. The reaction mixtures containing the radioactivesubstrate[1-³H]FPP (15-30 mCi/μmol) are incubated at 30.degree. C. for30 min and one volume of 1:1 (v/v) 40% aqueous KOH:methanol is added tothe suspension mixture. Liquid NaCl is added to saturate the solutionand 2 ml of naphtha containing 0.5% (v/v) squalene are likewise added.

The suspension is vortexed for 30 s. In each case 1 ml of the naphthalayer is applied to a packed 0.5.times.6 cm aluminum column (80-200mesh, Fisher) using a Pasteur pipette. The column has beenpre-equilibrated with 2 ml of naphtha containing 0.5% (v/v) squalene.The column is then eluted with 5.times.1 ml of toluene containing 0.5%(v/v) squalene. Squalene radioactivity is measured in Cytoscint (ICN)scintillation cocktail in a scintillation counter (Beckman).

This method is a modification of the method described in Radisky et al.,Biochemistry. 2000 Feb. 22; 39(7):1748-60, Zhang et al. (1993) Arch.Biochem. Biophys. 304, 133-143 and Poulter, C. D. et al. (1989) J. Am.Chem. Soc. 111, 3734-3739.

The sterol-acyltransferase activity can be determined as describedbelow:

A 200 ml main culture is inoculated at 1% strength from a 20 mlpreculture which has been incubated for two days and is incubated incomplete medium overnight. The cells are harvested and then washed intwo volumes of HP buffer (100 mM potassium phosphate buffer, pH 7.4; 0.5mM EDTA; 1 mM glutathione; 20 μM leupeptin; 64 μM benzamidine; 2 mMPMSF) and resuspended in HP buffer.

After adding 1 g of glass beads, the cells are disrupted by vortexing 8times for one minute each. The supernatant is ultracentrifuged at105000.times.g. The pellet is taken up in 1 ml of ACAT buffer (100 mMpotassium phosphate buffer pH7.4; 1 mM glutathione).

The enzyme assay is carried out in a volume of 500 μl. The substrateergosterol is taken up in 62.5 ml of 0.5.times.ACAT buffer with vigorousvortexing. 250 μl of this solution are used as substrate in the assay.To this, 20 μl of protein extract, 50 μl of water and 130 μl of0.5.times.ACAT buffer are added.

The mixture is incubated at 37.degree. C. for 15 min. Subsequently, 50μl of 14C-oleoyl-CoA (600000 dpm) are added and the reaction is stoppedafter one minute by adding 4 ml of chloroform/methanol (2:1). To this,500 μl of H₂O are added. The phases are separated by brieflycentrifuging the suspension at 2000.times.g. The lower phase isevaporated to dryness in a pear-shaped flask and redissolved in 100 μlof chloroform/methanol (4:1) and applied to a TLC plate (silica gel 60F254). The TLC is carried out using petroleum ether/diethyl ether/aceticacid 90:10:1 as eluent. The spots of the steryl ester fractions are cutout and the number of radioactive decays is determined in ascintillation column. The enzyme activity can be determined via theamount of sterile ester-bound 14C-oleoyl-CoA molecules.

In a preferred embodiment of the method of the invention7-dehydrocholesterol and/or the biosynthetic intermediates and/orintermediates thereof are prepared by culturing organisms, in particularyeasts, which have, compared to the wild type, an increased activity ofat least one of the activities selected from the group consisting ofΔ8-Δ7-isomerase activity, Δ5-desaturase activity and Δ24-reductaseactivity and which have additionally a reduced activity of at least oneof the activities selected from the group consisting ofC24-methyltransferase activity and Δ22-desaturase activity and whichhave additionally an increased HMG-CoA-reductase activity, an increasedlanosterol-C14-demethylase activity and an increased squalene-epoxidaseactivity.

In other preferred embodiments of the method of the invention,7-dehydrocholesterol and/or the biosynthetic intermediates and/orsecondary products thereof are prepared by culturing organisms, inparticular yeasts, which have, compared to the wild type,

an increased Δ8-Δ7-isomerase activity,

an increased Δ5-desaturase activity,

an increased Δ24-reductase activity,

an increased Δ8-Δ7-isomerase activity and an increased Δ5-desaturaseactivity,

an increased Δ8-Δ7-isomerase activity and an increased Δ24-reductaseactivity,

an increased Δ5-desaturase activity and an increased Δ24-reductaseactivity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity and an increased Δ24-reductase activity,

an increased Δ8-Δ7-isomerase activity and a reducedC24-methyltransferase activity,

an increased Δ5-desaturase activity and a reduced C24-methyltransferaseactivity,

an increased Δ24-reductase activity and a reduced C24-methyltransferaseactivity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity and a reduced C24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ24-reductaseactivity and a reduced C24-methyltransferase activity,

an increased Δ5-desaturase activity, an increased Δ24-reductase activityand a reduced C24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity and a reducedC24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity and a reduced, Δ22-desaturaseactivity,

an increased Δ5-desaturase activity and a reduced, Δ22-desaturaseactivity,

an increased Δ24-reductase activity and a reduced, Δ22-desaturaseactivity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity and a reduced, Δ22-desaturase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ24-reductaseactivity and a reduced, Δ22-desaturase activity,

an increased Δ5-desaturase activity, an increased Δ24-reductase activityand a reduced, Δ22-desaturase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity and a reducedΔ22-desaturase activity,

an increased Δ8-Δ7-isomerase activity, a reduced Δ22-desaturase activityand a reduced C24-ethyltransferase activity,

an increased Δ5-desaturase activity, a reduced Δ22-desaturase activityand a reduced C24-methyltransferase activity,

an increased Δ24-reductase activity, a reduced Δ22-desaturase activityand a reduced C24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, a reduced Δ22-desaturase activity and a reducedC24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ24-reductaseactivity, a reduced Δ22-desaturase activity and a reducedC24-methyltransferase activity,

an increased Δ5-desaturase activity, an increased Δ24-reductaseactivity, a reduced Δ22-desaturase activity and a reducedC24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, a reduced Δ22-desaturaseactivity and a reduced C24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity and an increased HMG-CoA-reductaseactivity,

an increased Δ5-desaturase activity and an increased HMG-CoA-reductaseactivity,

an increased Δ24-reductase activity and an increased HMG-CoA-reductaseactivity,

an increased Δ8-Δ7-isomerase activity, an increased HMG-CoA-reductaseactivity and an increased Δ5-desaturase activity,

an increased Δ8-Δ7-isomerase activity, an increased HMG-CoA-reductaseactivity and an increased Δ24-reductase activity,

an increased Δ5-desaturase activity, an increased HMG-CoA-reductaseactivity and an increased Δ24-reductase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased HMG-CoA-reductase activity and an increasedΔ24-reductase activity,

an increased Δ8-Δ7-isomerase activity, an increased HMG-CoA-reductaseactivity and a reduced C24-methyltransferase activity,

an increased Δ5-desaturase activity, an increased HMG-CoA-reductaseactivity and a reduced C24-methyltransferase activity,

an increased Δ24-reductase activity, an increased HMG-CoA-reductaseactivity and a reduced C24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased HMG-CoA-reductase activity and a reducedC24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ24-reductaseactivity, an increased HMG-CoA-reductase activity and a reducedC24-methyltransferase activity,

an increased Δ5-desaturase activity, an increased Δ24-reductase activityand a reduced C24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, an increasedHMG-CoA-reductase activity and a reduced C24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased HMG-CoA-reductaseactivity and a reduced, Δ22-desaturase activity,

an increased Δ5-desaturase activity, an increased HMG-CoA-reductaseactivity and a reduced, Δ22-desaturase activity,

an increased Δ24-reductase activity, an increased HMG-CoA-reductaseactivity and a reduced, Δ22-desaturase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased HMG-CoA-reductase activity and a reduced,Δ22-desaturase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ24-reductaseactivity, an increased HMG-CoA-reductase activity and a reduced,Δ22-desaturase activity,

an increased Δ5-desaturase activity, an increased Δ24-reductaseactivity, an increased HMG-CoA-reductase activity and a reduced,Δ22-desaturase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, an increasedHMG-CoA-reductase activity and a reduced Δ22-desaturase activity,

an increased Δ8-Δ7-isomerase activity, a reduced Δ22-desaturaseactivity, an increased HMG-CoA-reductase activity and a reducedC24-methyltransferase activity,

an increased Δ5-desaturase activity, a reduced Δ22-desaturase activity,an increased HMG-CoA-reductase activity and a reducedC24-methyltransferase activity,

an increased Δ24-reductase activity, a reduced Δ22-desaturase activity,an increased HMG-CoA-reductase activity and a reducedC24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, a reduced Δ22-desaturase activity, an increasedHMG-CoA-reductase activity and a reduced C24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ24-reductaseactivity, a reduced Δ22-desaturase activity, an increasedHMG-CoA-reductase activity and a reduced C24-methyltransferase activity,

an increased Δ5-desaturase activity, an increased Δ24-reductaseactivity, a reduced Δ22-desaturase activity, an increasedHMG-CoA-reductase activity and a reduced C24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, a reduced Δ22-desaturaseactivity, an increased HMG-CoA-reductase activity and a reducedC24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity and an increasedlanosterol-C14-demethylase activity,

an increased Δ5-desaturase activity and an increasedlanosterol-C14-demethylase activity,

an increased Δ24-reductase activity and an increasedlanosterol-C14-demethylase activity,

an increased Δ8-Δ7-isomerase activity, an increasedlanosterol-C14-demethylase activity and an increased Δ5-desaturaseactivity,

an increased Δ8-Δ7-isomerase activity, an increasedlanosterol-C14-demethylase activity and an increased Δ24-reductaseactivity,

an increased Δ5-desaturase activity, an increasedlanosterol-C14-demethylase activity and an increased Δ24-reductaseactivity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased lanosterol-C14-demethylase activity and anincreased Δ24-reductase activity,

an increased Δ8-Δ7-isomerase activity, an increasedlanosterol-C14-demethylase activity and a reduced C24-methyltransferaseactivity,

an increased Δ5-desaturase activity and a reduced C24-methyltransferaseactivity,

an increased Δ24-reductase activity, an increasedlanosterol-C14-demethylase activity and a reduced C24-methyltransferaseactivity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased lanosterol-C14-demethylase activity and a reducedC24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ24-reductaseactivity, an increased lanosterol-C14-demethylase activity and a reducedC24-methyltransferase activity,

an increased Δ5-desaturase activity, an increased Δ24-reductaseactivity, an increased lanosterol-C14-demethylase activity and a reducedC24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, an increasedlanosterol-C14-demethylase activity and a reduced C24-methyltransferaseactivity,

an increased Δ8-Δ7-isomerase activity, an increasedlanosterol-C14-demethylase activity and a reduced, Δ22-desaturaseactivity,

an increased Δ5-desaturase activity, an increasedlanosterol-C14-demethylase activity and a reduced, Δ22-desaturaseactivity,

an increased Δ24-reductase activity, an increasedlanosterol-C14-demethylase activity and a reduced, Δ22-desaturaseactivity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased lanosterol-C14-demethylase activity and areduced, Δ22-desaturase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ24-reductaseactivity, an increased lanosterol-C14-demethylase activity and areduced, Δ22-desaturase activity,

an increased Δ5-desaturase activity, an increased Δ24-reductaseactivity, an increased lanosterol-C14-demethylase activity and areduced, Δ22-desaturase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, an increasedlanosterol-C14-demethylase activity and a reduced, Δ22-desaturaseactivity,

an increased Δ8-Δ7-isomerase activity, a reduced, Δ22-desaturaseactivity, an increased lanosterol-C14-demethylase activity and a reducedC24-methyltransferase activity,

an increased Δ5-desaturase activity, a reduced, Δ22-desaturase activity,an increased lanosterol-C14-demethylase activity and a reducedC24-methyltransferase activity,

an increased Δ24-reductase activity, a reduced, Δ22-desaturase activity,an increased lanosterol-C14-demethylase activity and a reducedC24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, a reduced, Δ22-desaturase activity, an increasedlanosterol-C14-demethylase activity and a reduced C24-methyltransferaseactivity,

an increased Δ8-Δ7-isomerase activity, an increased Δ24-reductaseactivity, a reduced, Δ22-desaturase activity, an increasedlanosterol-C14-demethylase activity and a reduced C24-methyltransferaseactivity,

an increased Δ5-desaturase activity, an increased Δ24-reductaseactivity, a reduced, Δ22-desaturase activity, an increasedlanosterol-C14-demethylase activity and a reduced C24-methyltransferaseactivity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, a reduced, Δ22-desaturaseactivity, an increased lanosterol-C14-demethylase activity and a reducedC24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increasedlanosterol-C14-demethylase activity and an increased HMG-CoA-reductaseactivity,

an increased Δ5-desaturase activity, an increasedlanosterol-C14-demethylase activity and an increased HMG-CoA-reductaseactivity,

an increased Δ24-reductase activity, an increasedlanosterol-C14-demethylase activity and an increased HMG-CoA-reductaseactivity,

an increased AB-A7-isomerase activity, an increased HMG-CoA-reductaseactivity, an increased lanosterol-C14-demethylase activity and anincreased Δ5-desaturase activity,

an increased Δ8-Δ7-isomerase activity, an increased HMG-CoA-reductaseactivity, an increased lanosterol-C14-demethylase activity and anincreased Δ24-reductase activity,

an increased Δ5-desaturase activity, an increased HMG-CoA-reductaseactivity, an increased lanosterol-C14-demethylase activity and anincreased Δ24-reductase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased HMG-CoA-reductase activity, an increasedlanosterol-C14-demethylase activity and an increased Δ24-reductaseactivity,

an increased Δ8-Δ7-isomerase activity, an increased HMG-CoA-reductaseactivity, an increased lanosterol-C14-demethylase activity and a reducedC24-methyltransferase activity,

an increased Δ5-desaturase activity, an increased HMG-CoA-reductaseactivity, an increased lanosterol-C14-demethylase activity and a reducedC24-methyltransferase activity,

an increased Δ24-reductase activity, an increased HMG-CoA-reductaseactivity, an increased lanosterol-C14-demethylase activity and a reducedC24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased HMG-CoA-reductase activity, an increasedlanosterol-C14-demethylase activity and a reduced C24-methyltransferaseactivity,

an increased Δ8-Δ7-isomerase activity, an increased Δ24-reductaseactivity, an increased HMG-CoA-reductase activity, an increasedlanosterol-C14-demethylase activity and a reduced C24-methyltransferaseactivity,

an increased Δ5-desaturase activity, an increased Δ24-reductaseactivity, an increased lanosterol-C14-demethylase activity and a reducedC24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, an increasedHMG-CoA-reductase activity, an increased lanosterol-C14-demethylaseactivity and a reduced C24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased HMG-CoA-reductaseactivity, an increased lanosterol-C14-demethylase activity and a reducedΔ22-desaturase activity,

an increased Δ5-desaturase activity, an increased HMG-CoA-reductaseactivity, an increased lanosterol-C14-demethylase activity and a reducedΔ22-desaturase activity,

an increased Δ24-reductase activity, an increased HMG-CoA-reductaseactivity, an increased lanosterol-C14-demethylase activity and a reducedΔ22-desaturase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased HMG-CoA-reductase activity, an increasedlanosterol-C14-demethylase activity and a reduced Δ22-desaturaseactivity,

an increased Δ8-Δ7-isomerase activity, an increased Δ24-reductaseactivity, an increased HMG-CoA-reductase activity, an increasedlanosterol-C14-demethylase activity and a reduced Δ22-desaturaseactivity,

an increased Δ5-desaturase activity, an increased Δ24-reductaseactivity, an increased HMG-CoA-reductase activity and a reducedΔ22-desaturase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, an increasedHMG-CoA-reductase activity, an increased lanosterol-C14-demethylaseactivity and a reduced Δ22-desaturase activity,

an increased Δ8-Δ7-isomerase activity, a reduced Δ22-desaturaseactivity, an increased HMG-CoA-reductase activity, an increasedlanosterol-C14-demethylase activity and a reduced C24-methyltransferaseactivity,

an increased Δ5-desaturase activity, a reduced Δ22-desaturase activity,an increased HMG-CoA-reductase activity, an increasedlanosterol-C14-demethylase activity and a reduced C24-methyltransferaseactivity,

an increased Δ24-reductase activity, a reduced Δ22-desaturase activity,an increased HMG-CoA-reductase activity, an increasedlanosterol-C14-demethylase activity and a reduced C24-methyltransferaseactivity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, a reduced Δ22-desaturase activity, an increasedHMG-CoA-reductase activity, an increased lanosterol-C14-demethylaseactivity and a reduced C24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ24-reductaseactivity, a reduced Δ22-desaturase activity, an increasedHMG-CoA-reductase activity, an increased lanosterol-C14-demethylaseactivity and a reduced C24-methyltransferase activity,

an increased Δ5-desaturase activity, an increased Δ24-reductaseactivity, a reduced Δ22-desaturase activity, an increasedHMG-CoA-reductase activity, an increased lanosterol-C14-demethylaseactivity and a reduced C24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, a reduced Δ22-desaturaseactivity, an increased HMG-CoA-reductase activity,

an increased lanosterol-C14-demethylase activity and a reducedC24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, a reduced Δ22-desaturaseactivity, an increased HMG-CoA-reductase activity,

an increased lanosterol-C14-demethylase activity and a reducedC24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, a reduced Δ22-desaturaseactivity, an increased HMG-CoA-reductase activity,

an increased lanosterol-C14-demethylase activity, an increasedsqualene-epoxidase activity and a reduced C24-methyltransferaseactivity, or

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, a reduced Δ22-desaturaseactivity, an increased HMG-CoA-reductase activity, an increasedlanosterol-C14-demethylase activity, an increased squalene-epoxidaseactivity and a reduced C24-methyltransferase activity.

In further particularly preferred embodiments of the method of theinvention, 7-dehydrocholesterol and/or the biosynthetic intermediatesand/or secondary products thereof are prepared by culturing organisms,in particular yeasts, which have, compared to the wild type, anincreased Δ8-Δ7-isomerase activity, an increased Δ5-desaturase activity,an increased Δ24-reductase activity, a reduced Δ22-desaturase activity,an increased HMG-CoA-reductase activity, an increasedlanosterol-C14-demethylase activity and an increased squalene-epoxidaseactivity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, an increasedHMG-CoA-reductase activity, an increased lanosterol-C14-demethylaseactivity, an increased squalene-epoxidase activity and a reducedC24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, a reduced Δ22-desaturaseactivity, an increased HMG-CoA-reductase activity,

an increased lanosterol-C14-demethylase activity, an increasedsqualene-epoxidase activity and a reduced C24-methyltransferaseactivity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, a reduced Δ22-desaturaseactivity, an increased HMG-CoA-reductase activity,

an increased lanosterol-C14-demethylase activity, an increasedsqualene-epoxidase activity, an increased squalene-synthetase activityand a reduced C24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, a reduced Δ22-desaturaseactivity, an increased HMG-CoA-reductase activity,

an increased lanosterol-C14-demethylase activity, an increasedsqualene-epoxidase activity, an increased sterol-acyltransferaseactivity and a reduced C24-methyltransferase activity,

an increased Δ8-Δ7-isomerase activity, an increased Δ5-desaturaseactivity, an increased Δ24-reductase activity, a reduced Δ22-desaturaseactivity, an increased HMG-CoA-reductase activity, an increasedlanosterol-C14-demethylase activity, an increased squalene-epoxidaseactivity, an increased squalene-synthetase activity, an increasedsterol-acyltransferase activity and a reduced C24-methyltransferaseactivity.

Biosynthetic 7-dehydrocholesterol intermediates mean all compounds whichappear as intermediates during 7-dehydrocholesterol biosynthesis in theorganism used, preferably the compounds mevalonate, farnesylpyrophosphate, geraniol pyrophosphate, squalene epoxide,4-dimethylcholesta-8,14,24-trienol, 4,4-dimethylzymosterol, squalene,farnesol, geraniol, lanosterol, zymosterol, lathosterol,cholesta-7,24-dienol and cholesta-5,7,24-trienol.

Biosynthetic secondary products of zymosterol mean all compounds whichcan be derived biosynthetically from 7-dehydrocholesterol in theorganism used, i.e. for which 7-dehydrocholesterol appears as anintermediate. These may be compounds which the organism used producesnaturally from 7-dehydrocholesterol, such as, for example, cholesterolor vitamin D3 in mammals. However, they also mean compounds which can beproduced in the organism from 7-dehydrocholesterol only by introducinggenes and enzyme activities of other organisms for which the startingorganism has no orthologous gene.

It is possible, for example, to prepare secondary products from7-dehydrocholesterol, which are naturally present only in mammals, byintroducing mammalian genes into yeast:

Introducing a human or murine nucleic acid encoding a human or murineD-7-reductase enables the yeast to produce cholesterol.

Under UV irradiation, vitamin D₃ (cholecalciferol) is produced from7-dehydrocholesterol via provitamin D₃ by rearrangement.

Therefore, the biosynthetic secondary products of 7-dehydrocholesterolmean in particular provitamin D3, vitamin D₃ (cholecalciferol) and/orcholesterol.

Preferred biosynthetic secondary products are provitamin D₃ and inparticular vitamin D₃.

The compounds prepared in the method of the invention may be used inbiotransformations, chemical reactions and for therapeutic purposes, forexample for producing vitamin D₃ from 7-dehydrocholesterol via UVirradiation, or for producing steroid hormones via biotransformationstarting from cholesta-7,24-dienol or cholesta-5,7,24-trienol.

In the inventive method for preparing 7-dehydrocholesterol and/or thebiosynthetic intermediates and/or secondary products thereof, the stepof culturing the genetically modified organisms, also referred to astransgenic organisms hereinbelow, is preferably followed by harvestingsaid organisms and isolating 7-dehydrocholesterol and/or thebiosynthetic intermediates and/or secondary products thereof from saidorganisms.

The organisms are harvested in a manner known per se and appropriate forthe particular organism. Microorganisms such as bacteria, mosses, yeastsand fungi or plant cells which are cultured in liquid media byfermentation may be removed, for example, by centrifugation, decantingor filtration.

7-Dehydrocholesterol and/or the biosynthetic intermediates and/orsecondary products thereof are isolated from the harvested biomasstogether or each compound is harvested separately in a manner known perse, for example by extraction and, where appropriate, further chemicalor physical purification processes such as, for example, precipitationmethods, crystallography, thermal separation methods such asrectification methods or physical separation methods such as, forexample, chromatography.

The transgenic organisms, in particular yeasts, are preferably preparedeither by transforming the starting organisms, in particular yeasts,with a nucleic acid construct containing at least one nucleic acidselected from the group consisting of nucleic acids encoding aΔ8-Δ7-isomerase, nucleic acids encoding a Δ5-desaturase and nucleicacids encoding a Δ24-reductase which are functionally linked with one ormore regulatory signals ensuring transcription and translation inorganisms. In this embodiment, the transgenic organisms are preparedusing a nucleic acid construct.

In a particularly preferred embodiment, the above-described nucleic acidconstruct additionally contains at least one nucleic acid selected fromthe group consisting of nucleic acids encoding an HMG-CoA-reductaseactivity, nucleic acids encoding a lanosterol-C14-demethylase, nucleicacids encoding a squalene epoxidase, nucleic acids encoding a squalenesynthetase and nucleic acids encoding a sterol acyltransferase which arefunctionally linked to one or more regulatory signals ensuringtranscription and translation in organisms.

However, the transgenic organisms may also preferably be prepared bytransforming the starting organisms, in particular yeasts, with at leastone nucleic acid construct selected from the group consisting of nucleicacid constructs containing nucleic acids encoding a Δ8-Δ7-isomerase,nucleic acid construct containing nucleic acids encoding a Δ5-desaturaseand nucleic acid construct containing nucleic acids encoding aΔ24-reductase which nucleic acids are in each case functionally linkedto one or more regulatory signals ensuring transcription and translationin organisms. In this embodiment, the transgenic organisms are preparedusing individual nucleic acid constructs or a combination of nucleicacid constructs.

In a particularly preferred embodiment, the above-described combinationof nucleic acid constructs additionally comprises at least one nucleicacid construct selected from the group consisting of nucleic acidconstruct containing nucleic acids encoding an HMG-CoA-reductaseactivity, nucleic acid construct containing nucleic acids encoding alanosterol-C14-demethylase, nucleic acid construct containing nucleicacids encoding a squalene epoxidase, nucleic acid construct containingnucleic acids encoding a squalene synthetase and nucleic acid constructcontaining nucleic acids encoding a sterol acyltransferase which nucleicacids are in each case functionally linked to one or more regulatorysignals ensuring transcription and translation in organisms.

Nucleic acid constructs in which the encoding nucleic acid sequence isfunctionally linked to one or more regulatory signals ensuringtranscription and translation in organisms, in particular in yeasts, arealso referred to as expression cassettes hereinbelow.

Examples of nucleic acid constructs containing said expression cassetteare vectors and plasmids.

Accordingly, the invention further relates to nucleic acid constructs,in particular nucleic acid constructs functioning as expressioncassettes, which contain at least one nucleic acid selected from thegroup consisting of nucleic acids encoding a Δ8-Δ7-isomerase, nucleicacids encoding a Δ5-desaturase and nucleic acids encoding aΔ24-reductase which are functionally linked to one or more regulatorysignals ensuring transcription and translation in organisms.

In a preferred embodiment, said nucleic acid construct additionallycomprises at least one nucleic acid selected from the group consistingof nucleic acids encoding an HMG-CoA-reductase activity, nucleic acidsencoding a lanosterol-C14-demethylase, nucleic acids encoding a squaleneepoxidase, nucleic acids encoding a squalene synthetase and nucleicacids encoding a sterol acyltransferase which are functionally linked toone or more regulatory signals ensuring transcription and translation inorganisms.

As an alternative, it is also possible to prepare the transgenicorganisms of the invention by transformation with individual nucleicacid constructs or with a combination of nucleic acid constructs, saidcombination comprising at least one nucleic acid construct selected fromthe groups A to C

A nucleic acid construct comprising nucleic acids encoding aΔ8-Δ7-isomerase, which are functionally linked to one or more regulatorysignals ensuring transcription and translation in organisms,

B nucleic acid construct comprising nucleic acids encoding aΔ5-desaturase, which are functionally linked to one or more regulatorysignals ensuring transcription and translation in organisms and

C nucleic acid construct comprising nucleic acids encoding aΔ24-reductase, which are functionally linked to one or more regulatorysignals ensuring transcription and translation in organisms, and atleast one nucleic acid construct selected from the groups D to H

D nucleic acid construct comprising nucleic acids encoding an HMG-CoAreductase, which are functionally linked to one or more regulatorysignals ensuring transcription and translation in organisms, E nucleicacid construct comprising nucleic acids encoding a lanosterolC14-demethylase, which are functionally linked to one or more regulatorysignals ensuring transcription and translation in organisms, F nucleicacid construct comprising nucleic acids encoding a squalene epoxidase,which are functionally linked to one or more regulatory signals ensuringtranscription and translation in organisms, G nucleic acid constructcomprising nucleic acids encoding a squalene synthetase, which arefunctionally linked to one or more regulatory signals ensuringtranscription and translation in organisms, H nucleic acid constructcomprising nucleic acids encoding a sterol acyltransferase, which arefunctionally linked to one or more regulatory signals ensuringtranscription and translation in organisms.

The regulatory signals preferably contain one or more promoters whichensure transcription and translation in organisms, in particular inyeasts.

The expression cassettes include regulatory signals, i.e. regulatorynucleic acid sequences, which control expression of the coding sequencein the host cell. According to a preferred embodiment, an expressioncassette comprises upstream, i.e. at the 5′ end of the coding sequence,a promoter and downstream, i.e. at the 3′ end, a terminator and, whereappropriate, further regulatory elements which are operatively linked tothe coding sequence for at least one of the above-described geneslocated in between.

Operative linkage means the sequential arrangement of promoter, codingsequence, where appropriate, terminator and, where appropriate, furtherregulatory elements in such a way that each of the regulatory elementscan properly carry out its function in the expression of the codingsequence.

The preferred nucleic acid constructs, expression cassettes and plasmidsfor yeasts and fungi and methods for preparing transgenic yeasts andalso the transgenic yeasts themselves are described by way of examplebelow.

A suitable promoter of the expression cassette is in principle anypromoter which is able to control the expression of foreign genes inorganisms, in particular in yeasts.

Preference is given to using in particular a promoter which is subjectto reduced regulation in yeast, such as, for example, the medium ADHpromoter.

This promoter fragment of the ADH12s promoter, also referred to as ADH1hereinbelow, exhibits nearly constitutive expression (Ruohonen L,Penttila M, Keranen S. (1991) Optimization of Bacillus α-amylaseproduction by Saccharomyces cerevisiae. Yeast. May-June; 7(4):337-462;Lang C, Looman A C. (1995) Efficient expression and secretion ofAspergillus niger RH5344 polygalacturonase in Saccharomyces cerevisiae.Appl Microbiol Biotechnol. December; 44(1-2):147-56) so thattranscriptional regulation no longer proceeds via intermediates ofergosterol biosynthesis.

Other preferred promoters with reduced regulation are constitutivepromoters such as, for example, the yeast TEF1 promoter, the yeast GPDpromoter or the yeast PGK promoter (Mumberg D, Muller R, Funk M. (1995)Yeast vectors for the controlled expression of heterologous proteins indifferent genetic backgrounds. Gene. 1995 Apr. 14; 156(1):119-22; Chen CY, Oppermann H, Hitzeman R A. (1984) Homologous versus heterologous geneexpression in the yeast, Saccharomyces cerevisiae. Nucleic Acids Res.December 11; 12(23):8951-70).

The expression cassette may also contain inducible promoters, inarticular a chemically inducible promoter which can be used to controlexpression of the nucleic acids encoding a Δ8-Δ7-isomerase,Δ5-desaturase, Δ24-reductase, HMG-CoA-reductase,lanosterol-C14-demethylase, squalene epoxidase, squalene synthetase orsterol acyltransferase in the organism at a particular time.

Promoters of this kind, such as, for example, the yeast Cupl promoter(Etcheverry T. (1990) Induced expression using yeast coppermetallothionein promoter. Methods Enzymol. 1990; 185:319-29), the yeastGall-10 promoter (Ronicke V, Graulich W, Mumberg D, Muller R, Funk M.(1997) Use of conditional promoters for expression of heterologousproteins in Saccharomyces cerevisiae, Methods Enzymol. 283:313-22) orthe yeast Pho5 promoter (Bajwa W, Rudolph H, Hinnen A. (1987) PHO5upstream sequences confer phosphate control on the constitutive PHO3gene. Yeast. 1987 March; 3(1):33-42), may be used, for example.

A suitable terminator of the expression cassette is in principle anyterminator which is able to control the expression of foreign genes inorganisms, in particular in yeasts.

Preference is given to the tryptophan terminator of yeasts (TRP1terminator).

An expression cassette is preferably prepared by fusing a suitablepromoter with the above-described nucleic acids encoding aΔ8-Δ7-isomerase, Δ5-desaturase, Δ24-reductase, HMG-CoA-reductase,lanosterol-C14-demethylase, squalene epoxidase, squalene synthetase orsterol acyltransferase and, where appropriate, a terminator according tocommon recombination and cloning techniques as described, for example,in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1989) and in T. J. Silhavy, M. L. Berman and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984) and in Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Greene Publishing Assoc. andWiley-Interscience (1987).

The nucleic acids of the invention may be prepared synthetically orobtained naturally or may contain a mixture of synthetic and naturalnucleic acid components and may also comprise various heterologous genesections of various organisms.

As described above, preference is given to synthetic nucleotidesequences with codons which are preferred by yeasts. These codons whichare preferred by yeasts may be determined from codons which have thehighest frequency in proteins and which are expressed in most of theinteresting yeast species.

When preparing an expression cassette, it is possible to manipulatevarious DNA fragments in order to obtain a nucleotide sequence whichexpediently can be read in the correct direction and is provided with acorrect reading frame. The DNA fragments may be linked to one another byattaching adaptors or linkers to said fragments.

Expediently, the promoter and terminator regions may be provided in thedirection of transcription with a linker or polylinker which containsone or more restriction sites for inserting this sequence. Normally, thelinker has from 1 to 10, mostly from 1 to 8, preferably from 2 to 6,restriction sites. Generally, the linker is, within the regulatoryregions, less than 100 bp, frequently less than 60 bp, but at least 5bp, in length. The promoter may be both native or homologous andnon-native or heterologous to the host organism. The expression cassettepreferably includes in the 5′-3′ direction of transcription thepromoter, a coding nucleic acid sequence or a nucleic acid construct anda region for transcriptional termination. Various termination regionscan be exchanged with one another randomly.

It is furthermore possible to use manipulations which provideappropriate restriction cleavage sites or which remove excess DNA orrestriction cleavage sites. In those cases for which insertions,deletions or substitutions such as, for example, transitions andtransversions are suitable, in vitro mutagenesis, primer repair,restriction or ligation can be used.

In suitable manipulations such as, for example, restriction,“chewing-back” or filling-in of protruding ends to form “blunt ends”,complementary fragment ends may be provided for ligation.

The invention further relates to the use of the above-described nucleicacids, the above-described nucleic acid constructs or theabove-described proteins for preparing transgenic organisms, inparticular yeasts.

Preferably, said transgenic organisms, in particular yeasts, have anincreased content of 7-dehydrocholesterol and/or of the biosyntheticintermediates and/or secondary products thereof compared to the wildtype.

Therefore, the invention further relates to the use of theabove-described nucleic acids or the nucleic acid constructs of theinvention for increasing the content of 7-dehydrocholesterol and/or ofthe biosynthetic intermediates and/or secondary products thereof inorganisms.

The above-described proteins and nucleic acids may be used for producing7-dehydrocholesterol and/or the biosynthetic intermediates and/orsecondary products thereof in transgenic organisms.

The transfer of foreign genes into the genome of an organism, inparticular of yeast, is referred to as transformation.

For this purpose, methods known per se can be used for transformation,in particular in yeasts.

Examples of suitable methods for transforming yeasts are the LiAC methodas described in Schiestl R H, Gietz R D. (1989) High efficiencytransformation of intact yeast cells using single stranded nucleic acidsas a carrier, Curr Genet. December; 16 (5-6):339-46, electroporation asdescribed in Manivasakam P, Schiestl R H. (1993) High efficiencytransformation of Saccharomyces cerevisiae by electroporation. NucleicAcids Res. September 11; 21(18):4414-5, and the preparation ofprotoplasts, as described in Morgan A J. (1983) Yeast strain improvementby protoplast fusion and transformation, Experientia Suppl. 46:155-66

The construct to be expressed is preferably cloned into a vector, inparticular into plasmids which are suitable for transforming yeasts,such as, for example, the vector systems Yep24 (Naumovski L, Friedberg EC (1982) Molecular cloning of eucaryotic genes required for excisionrepair of UV-irradiated DNA: isolation and partial characterization ofthe RAD3 gene of Saccharomyces cerevisiae. J Bacteriol October;152(1):323-31), Yep13 (Broach J R, Strathern J N, Hicks J B. (1979)Transformation in yeast: development of a hybrid cloning vector andisolation of the CAN1 gene. Gene. 1979 December; 8(1):121-33), the pRSseries of vectors (Centromer and Episomal) (Sikorski R S, Hieter P.(1989) A system of shuttle vectors and yeast host strains designed forefficient manipulation of DNA in Saccharomyces cerevisiae. Genetics.May; 122(1):19-27) and the vector systems YCp19 or pYEXBX.

Accordingly, the invention furthermore relates to vectors, in particularplasmids, which comprise the above-described nucleic acids, nucleic acidconstructs or expression cassettes.

The invention further relates to a method for preparing geneticallymodified organisms by functionally introducing an above-describednucleic acid or an above-described nucleic acid construct into thestarting organism.

The invention further relates to said genetically modified organisms,the genetic modification increasing at least one of the activitiesselected from the group consisting of Δ8-Δ7-isomerase activity,Δ5-desaturase activity and Δ24-reductase activity, compared to a wildtype.

Preferably, at least one of the activities is increased by increasingthe gene expression of at least one nucleic acid selected from the groupconsisting of nucleic acids encoding a Δ8-Δ7-isomerase, nucleic acidsencoding a Δ5-desaturase and nucleic acids encoding a Δ24-reductase.

Preferably, gene expression of the above-described nucleic acids isincreased by increasing in the organism the copy number of the nucleicacids encoding a Δ8-Δ7-isomerase, encoding a Δ5-desaturase and/orencoding a Δ24-reductase.

Accordingly, the invention preferably relates to an above-describedgenetically modified organism which contains two or more nucleic acidsencoding a Δ8-Δ7-isomerase and/or two or more nucleic acids encoding aΔ5-desaturase and/or two or more nucleic acids encoding a Δ24-reductase.

In a preferred embodiment, the genetically modified organism has,compared to the wild type, in addition to the above-described geneticmodifications a reduced activity of at least one of the activitiesselected from the group consisting of C24-methyltransferase activity andΔ22-desaturase activity.

The reduction of at least one of the activities is preferably caused byreducing, compared to the wild type, gene expression of at least onenucleic acid selected from the group consisting of nucleic acidsencoding a C24-methyltransferase and nucleic acids encoding aΔ22-desaturase.

A particularly preferred genetically modified organism has, apart fromthe above-described genetic modifications, no functionalC24-methyltransferase gene and/or Δ22-desaturase gene.

Particular preference is given to above-mentioned genetically modifiedorganisms in which the genetic modification additionally increases atleast one of the activities selected from the group consisting ofHMG-CoA-reductase activity, lanosterol-C14-demethylase activity,squalene-epoxidase activity, squalene-synthetase activity andsterol-acyltransferase activity compared to a wild type.

Preferably, at least one of these activities is increased, as mentionedabove, by increasing, compared to the wild type, gene expression of atleast one nucleic acid selected from the group consisting of nucleicacids encoding an HMG-CoA-reductase activity, nucleic acids encoding alanosterol-C14-demethylase, nucleic acids encoding a squalene epoxidase,nucleic acids encoding a squalene synthetase and nucleic acids encodinga sterol acyltransferase.

Preferably, gene expression of at least one nucleic acid selected fromthe group consisting of nucleic acids encoding an HMG-CoA-reductaseactivity, nucleic acids encoding a lanosterol-C14-demethylase, nucleicacids encoding a squalene epoxidase, nucleic acids encoding a squalenesynthetase and nucleic acids encoding a sterol acyltransferase isincreased compared to the wild type by increasing in the organism thecopy number of at least one nucleic acid selected from the groupconsisting of nucleic acids encoding an HMG-CoA-reductase activity,nucleic acids encoding a lanosterol-C14-demethylase, nucleic acidsencoding a squalene epoxidase, nucleic acids encoding a squalenesynthetase and nucleic acids encoding a sterol acyltransferase.

Accordingly, the invention preferably relates to an above-describedgenetically modified organism which contains two or more of at least onenucleic acid selected from the group consisting of nucleic acidsencoding an HMG-CoA-reductase activity, nucleic acids encoding alanosterol-C14-demethylase, nucleic acids encoding a squalene epoxidase,nucleic acids encoding a squalene synthetase and nucleic acids encodinga sterol acyltransferase.

In particular, the invention preferably relates to a geneticallymodified organism which contains, in addition to the above-describedgenetic modifications, two or more nucleic acids encoding anHMG-CoA-reductase and/or two or more nucleic acids encoding alanosterol-C14-demethylase and/or two or more nucleic acids encoding asqualene epoxidase and/or two or more nucleic acids encoding a squalenesynthetase and/or two or more nucleic acids encoding a sterolacyltransferase.

The above-described genetically modified organisms have, compared to thewild type, an increased content of 7-dehydrocholesterol and/or of thebiosynthetic intermediates and/or secondary products thereof.

Accordingly, the invention relates to an above-described geneticallymodified organism which, compared to the wild type, has an increasedcontent of 7-dehydrocholesterol and/or of the biosynthetic intermediatesand/or secondary products thereof.

Preferred genetically modified organisms are yeasts or fungi which havebeen genetically modified according to the invention, in particularyeasts which have been genetically modified according to the invention,in particular the yeast species Saccharomyces cerevisiae which has beengenetically modified according to the invention, in particular thegenetically modified yeast strains Saccharomyces cerevisiae AH22,Saccharomyces cerevisiae GRF, Saccharomyces cerevisiae DBY747 andSaccharomyces cerevisiae BY4741.

In the scope of the present invention, increasing the content of7-dehydrocholesterol and/or of the biosynthetic intermediates and/orsecondary products thereof preferably means the artificially acquiredability to produce biosynthetically an increased amount of at least oneof these compounds mentioned above in the genetically modified organismcompared to the genetically unmodified organism.

Accordingly, as mentioned at the beginning, wild type preferably meansthe genetically unmodified organism, but in particular the referenceorganism mentioned above.

An increased content of 7-dehydrocholesterol and/or of the biosyntheticintermediates and/or secondary products thereof in comparison with thewild type means in particular the increase in the content of at leastone of the abovementioned compounds in the organism by at least 50%,preferably 100%, more preferably 200%, particularly preferably 400%, incomparison with the wild type.

The content of at least one of the mentioned compounds is preferablydetermined according to analytical methods known per se and preferablyrefers to those compartments of the organism, in which sterols areproduced.

The invention is illustrated by the following examples but is notlimited to them:

I. GENERAL EXPERIMENTAL CONDITIONS 1. Restriction

Restriction of the plasmids (1 to 10 μg) was carried out in 30 μlreaction mixtures. For this purpose, the DNA was taken up in 24 μl ofH₂0 and admixed with 3 μl of the appropriate buffer, 1 ml of BSA (bovineserum albumin) and 2 μl of enzyme. The enzyme concentration was 1unit/μl or 5 units/μl, depending on the amount of DNA. In some cases, 1μl of RNase was added to the reaction mixture in order to degrade thetRNA. The restriction mixture was incubated at 37.degree. C. for 2hours. The restriction was monitored using a minigel.

2. Gel Electrophoreses

The gel electrophoreses were carried out in minigel or wide minigelapparatuses. The minigels (approx. 20 ml, 8 pockets) and the wideminigels (50 ml, 15 or 30 pockets) consisted of 1% strength agarose inTAE. The running buffer used was 1.times.TAE.

After adding 3 μl of stop solution, the samples (10 μl) were applied.λ-DNA cut with Hindlll (bands at: 23.1 kb; 9.4 kb; 6.6 kb; 4.4 kb; 2.3kb; 2.0 kb; 0.6 kb) served as standard. For fractionation, a voltage of80 V was applied for 45 to 60 min. Thereafter, the gel was stained inethidium bromide solution and documented under UV light using the INTASvideo documentation system or photographed using an orange filter.

3. Gel Elution

The desired fragments were isolated by means of gel elution. Therestriction mixture was applied to several pockets of a minigel andfractionated. Only A-Hindlll and a “sacrifice lane” were stained inethidium bromide solution, examined under UV light, and the desiredfragment was marked. This prevented the DNA of the remaining pocketsfrom being damaged by ethidium bromide and UV light. Putting the stainedand unstained gel slices side by side made it possible to excise thedesired fragment from the unstained gel slice on the basis of themarking. The agarose slice with the fragment to be isolated wasintroduced into a dialysis tube, sealed in air-bubble-free together witha small amount of TAE buffer and introduced into the BioRad minigelapparatus. The running buffer was 1.times.TAE and the voltage was 100 Vfor 40 min. Afterward, the polarity was switched for 2 min in order toredissolve DNA sticking to the dialysis tube. The buffer in the dialysistube, which contained the DNA fragments, was transferred to reactionvessels and subjected to ethanol precipitation. For this purpose, 1/10volume of 3M sodium acetate, tRNA (1 μl per 50 μl of solution) and 2.5volumes of ice-cold 96% strength ethanol were added to the DNA solution.The mixture was incubated at −20.degree. C. for 30 min and then removedby centrifugation at 12 000 rpm, 4.degree. C., 30 min. The DNA pelletwas dried and taken up in 10 to 50 μl of H₂0 (depending on the amount ofDNA).

4. Klenow Treatment

The Klenow treatment fills in protruding ends of DNA fragments,resulting in blunt ends. Per 1 μg of DNA, the following reaction mixturewas pipetted: DNA .times. .times. pellet+.times. 11 .times. μ .times..times. 1 .times. .times. H 2 .times. 0+.times. 1.5 .times. .times. 10.times. Klenow .times. .times. buffer+.times. 1 .times. μ .times..times. I .times. .times. 0.1 .times. .times. M .times. .times.DTT+.times. 1 .times. μ .times. .times. I .times. .times. nucleotide.times. .times. (dNTP .times. .times. 2 .times. .times. mM)+.times. 1.times. μ .times. .times. I .times. .times. Klenow .times. .times.polymerase .times. .times. (1.times. .times. unit/μ .times. .times. I)25

The DNA should be from an ethanol precipitation, in order to preventcontaminations from inhibiting the Klenow polymerase. The reactionmixture was incubated at 37.degree. C. for 30 min, and the reaction wasstopped by incubating for another 5 min at 70.degree. C. The DNA wasrecovered from the reaction mixture by ethanol precipitation and takenup in 10 μl of H₂0.

5. Ligation

The DNA fragments to be ligated were combined. The final volume of 13.1μl contained approx. 0.5 μl of DNA with a vector/insert ratio of 1:5.The sample was incubated at 70.degree. C. for 45 seconds, cooled to roomtemperature (approx. 3 min) and then incubated on ice for 10 min. Theligation buffers were then added: 2.6 μl of 500 mM Tris-HCl pH 7.5 and1.3 μl of 100 mM MgCl₂, followed by incubation on ice for a further 10min. After adding 1 μl of 500 mM DTT and 1 μl of 10 mM ATP and another10 min on ice, 1 μl of ligase (1 unit/μl) was added. The whole treatmentshould be carried out as free from vibrations as possible so thatadjoining DNA ends are not separated again. The ligation was carried outat 14.degree. C. over night.

6. Transformation of E. coli

Competent Escherichia coli (E. coli) NM522 cells were transformed withthe DNA of the ligation mixture. A reaction mixture containing 50 μg ofthe pScL3 plasmids and a reaction mixture without DNA were run aspositive control and zero control, respectively. For each transformationmixture, 100 μl of 8% PEG solution, 10 μl of DNA and 200 μl of competentcells (E. coli NM522) were pipetted into a benchtop-centrifuge tube. Thereaction mixtures were put on ice for 30 min and agitated occasionally.

Then the heat shock was carried out: 1 min at 42.degree. C. Forregeneration, 1 ml of LB medium was added to the cells and thesuspension was incubated on a shaker at 37.degree. C. for 90 min. Ineach case, 100 μl of the undiluted reaction mixtures, a 1:10 dilutionand a 1:100 dilution were plated on LB+ampicillin plates and incubatedat 37.degree. C. over night.

7. Plasmid Isolation from E. coli (Miniprep)

E. coli colonies were grown in 1.5 ml of LB+ampicillin medium inbenchtop-centrifuge tubes at 37.degree. C. and 120 rpm over night. Onthe next day, the cells were removed by centrifugation at 5000 rpm and4.degree. C. for 5 min and the pellet was taken up in 50 μl of TEbuffer. 100 μl of 0.2 N NaOH, 1% SDS solution were added to and mixedwith each reaction mixture, and the mixture was put on ice for 5 min(lysis of the cells). Then, 400 μl of Na acetate/NaCl solution (230 μlof H₂0, 130 μl of 3 M sodium acetate, 40 μl of 5M NaCl) were added, thereaction mixture was mixed and put on ice for a further 15 min (proteinprecipitation). After centrifugation at 11 000 rpm for 15 minutes, thesupernatant containing the plasmid DNA was transferred to an Eppendorfvessel. If the supernatant was not completely clear, centrifugation wasrepeated. 360 μl of ice-cold isopropanol were added to the supernatantand the reaction mixture was incubated at −20.degree. C. for 30 min (DNAprecipitation). The DNA was removed by centrifugation (15 min, 12 000rpm, 4.degree. C.), the supernatant was discarded, the pellet was washedin 100 μl of ice-cold 96% strength ethanol, incubated at −20.degree. C.for 15 min and again removed by centrifugation (15 min, 12 000 rpm,4.degree. C.). The pellet was dried in a Speed Vac and then taken up in100 μl of H₂0. The plasmid DNA was characterized by restrictionanalysis. For this purpose, 10 μl of each reaction mixture wererestriction-digested and fractionated gel-electrophoretically in a wideminigel (see above).

8. Plasmid Preparation from E. coli (Maxiprep)

In order to isolate larger amounts of plasmid DNA, the maxiprep methodwas carried out. Two flasks with 100 ml of LB+ampicillin medium wereinoculated with a colony or with 100 μl of a frozen culture whichcarries the plasmid to be isolated and incubated at 37.degree. C. and120 rpm over night. On the next day, the culture (200 ml) wastransferred to a GSA beaker and centrifuged at 4000 rpm (2600.times.g)for 10 min. The cell pellet was taken up in 6 ml of TE buffer. The cellwall was digested by adding 1.2 ml of lysozyme solution (20 mg/ml of TEbuffer) and incubated at room temperature for 10 min. Subsequently, thecells were lysed with 12 ml of a 0.2 N NaOH, 1% SDS solution, followedby incubation at room temperature for another 5 min. The proteins wereprecipitated by adding 9 ml of a cooled 3 M sodium acetate solution (pH4.8) and incubation on ice for 15 minutes. After centrifugation (GSA: 13000 rpm (27 500.times.g), 20 min, 4.degree. C.), the supernatantcontaining the DNA was transferred to a new GSA beaker and the DNA wasprecipitated with 15 ml of ice-cold isopropanol and incubation at−20.degree. C. for 30 min. The DNA pellet was washed in 5 ml of ice-coldethanol and dried in air (approx. 30-60 min). Thereafter, it was takenup in 1 ml of H₂O. The plasmid was checked by restriction analysis. Theconcentration was determined by applying dilutions to a minigel. Thesalt content was reduced by microdialysis (pore size 0.025 μm) for 30-60minutes.

9. Transformation of Yeast

For the transformation of yeast, a preculture of the strainSaccharomyces cerevisiae AH22 was prepared. A flask containing 20 ml ofYE medium was inoculated with 100 μl of the frozen culture and incubatedat 28.degree. C. and 120 rpm over night. The main culture was carriedout under the same conditions in flasks containing 100 ml of YE mediumwhich was inoculated with 10 μl, 20 μl or 50 μl of the preculture.

9.1 Preparation of Competent Cells

On the next day, the cells in the flasks were counted by means of aThoma chamber and the flask containing from 3-5.times.10⁷ cells/ml waschosen for the subsequent procedure. The cells were harvested bycentrifugation (GSA: 5000 rpm (4000.times.g) 10 min). The cell pelletwas taken up in 10 ml of TE buffer and distributed into twobenchtop-centrifuged tubes (5 ml each). The cells were removed bycentrifugation at 6000 rpm for 3 min and then washed twice with in eachcase 5 ml of TE buffer. The cell pellet was then taken up in 330 μl oflithium acetate buffer per 10⁹ cells, transferred to a sterile 50 mlErlenmeyer flask and agitated at 28.degree. C. for one hour. As aresult, the cells were competent for transformation.

9.2 Transformation

For each transformation mixture, 15 μl of herring sperm DNA (10 mg/ml),10 μl of the DNA to be transformed (approx. 0.5 μg) and 330 μl ofcompetent cells were pipetted into a benchtop-centrifuged tube andincubated at 28.degree. C. for 30 min (without agitation). Then, 700 μlof 50% PEG 6000 were added and the suspension was incubated at28.degree. C. for another hour, without agitation. This was followed bya heat shock at 42.degree. C. for 5 min. 100 μl of the suspension wereplated on selection medium (YNB, Difco) in order to select for leucineprototrophy. In the case of selection for G418 resistance, the cells areregenerated after the heat shock (see under 9.3 Regeneration phase).

9.3 Regeneration Phase

Since the selection marker is the resistance to G418, the cells neededtime to express the resistance gene. 4 ml of YE medium were added to thetransformation mixtures which were then incubated on the shaker (120rpm) at 28.degree. C. over night. On the next day, the cells wereremoved by centrifugation (6000 rpm, 3 min), taken up in 1 ml YE medium,and 100 μl or 200 μl thereof were plated on YE+G418 plates. The plateswere incubated at 28.degree. C. for several days.

10. PCR Reaction Conditions

The reaction conditions for the polymerase chain reaction must beoptimized in each individual case and do not apply absolutely to eachreaction mixture. Thus it is possible, inter alia, to vary the amount ofDNA used, the salt concentrations and the melting temperature. For ourtask, it proved advantageous to combine in an Eppendorf vessel which wassuitable for use in a thermocycler the following substances: 5 μl ofSuper buffer, 811 of dNTPs (0.625 μM each), 5′ primer, 3′ primer and 0.2μg of template DNA, dissolved in enough water so as to result in a totalvolume of 50 μl for the PCR reaction mixture, were added to 2 μl of(=0.1 U) Super Taq polymerase. The reaction mixture was brieflycentrifuged and overlaid with a drop of oil. Between 37 and 40 cycleswere chosen for amplification.

II. EXAMPLES Example 1

Expression and overexpression of a truncated HMG-CoA reductase, asqualene epoxidase (ERG1) and/or a lanosterol-C14-demethylase (ERG11),partially with deletion of ERG5 and ERG6 in S. cerevisiae GRF18 andGRFura3, respectively.

1.1 Preparation of the Plasmids pFlat1 and pFlat3 and pFlat4

The expression vector pFlat3 was prepared by linearizing the plasmidYEp24 (Naumovski L, Friedberg E C (1982) Molecular cloning of eucaryoticgenes required for excision repair of UV-irradiated DNA: isolation andpartial characterization of the RAD3 gene of Saccharomyces cerevisiae. JBacteriol October; 152(1):323-31) via restriction with Sphl and a 900 bySphl fragment of the vector pPT2B (Lang C, Looman A C. (1995) Efficientexpression and secretion of Aspergillus niger RH5344 polygalacturonasein Saccharomyces cerevisiae. Appl Microbiol Biotechnol. December;44(1-2): 147-56) which contains the ADH1 promoter and the TRP1terminator of the yeast Saccharomyces cerevisiae and a multiple-cloningsite of the vector pUC19 (Yanisch-Perron C, Vieira J, Messing J. (1985)Improved M13 phage cloning vectors and host strains: nucleotidesequences of the M13 mp18 and pUC19 vectors. Gene. 1985; 33(1): 103-19.)was integrated.

The multiple-cloning site was extended by a polylinker containing therestriction sites Notl and Xhol. The polylinker was integrated via theSall cleavage site of the vector. The resulting plasmid is denotedpFlat1.

The vector pFlat3 was prepared by linearizing the vector pFlat1 by theenzyme Ncol and blunt-ending it by means of Klenow treatment. This wasfollowed by integrating a BamHl fragment which had been blunt-ended bymeans of Klenow-polymerase treatment and which contains the yeast LEU2gene and originates from the plasmid YDpL (Berben, G., Dumont, J.,Gilliquet, V., Bolle, P. A. and Hilger F. (1991) The YDp Plasmids: aUniform Set of Vectors Bearing Versatile Disruption Cassettes forSaccharomyces cerevisiae. Yeast 7: 475-477).

The vector pFlat4 was prepared by linearizing the vector pFlat1 by theenzyme Ncol and blunt-ending it by means of Klenow treatment. This wasfollowed by integrating a BamHl fragment which had been blunt-ended bymeans of Klenow-polymerase treatment and which contains the yeast HIS3gene and originates from plasmid YDpH (Berben et al., 1991).

1.2 Integration of ERG1, ERG11, ERG4, ERG2 or ERG3 or of theΔ24-Reductase Gene into the Vectors pFLat1, pFlat3 and pFlat4

First, a Notl restriction cleavage site was inserted at the 5′-codingside of the genes ERG1, ERG11, ERG4, Δ24-reductase, ERG2 or ERG3 and anXhol restriction cleavage site was inserted at the 3′-coding side ofsaid genes by means of PCR and the corresponding coding regions wereamplified. Subsequently, the amplicons were treated with the restrictionenzymes Notl and Xhol. The plasmids pFlat1, pFlat3 and pFlat4 weretreated in parallel with enzymes Notl and Xhol. The cleaved ampliconswere then integrated into the cleaved plasmids via ligation using T4ligase. FIG. 7 depicts as an example the plasmid pFLAT-3-ERG4.

Primer sequences for cloning ERG1, ERG11, ERG2, ERG3, ERG4,Δ24-reductase: TABLE-US-00002 Primer ERG1-5′ (SEQ. ID. No. 51):CTGCGGCCGC ATCATGTCTG CTGTTAACGT TGC Primer ERG1-3′ (SEQ. ID. No. 52):TTCTCGAGTT AACCAATCAA CTCACCAAAC Primer ERG11-5′ (SEQ. ID. No. 53):CTGCGGCCGCAGGATGTCTGCTACCAAGTCAATCG Primer ERG11-3′ (SEQ. ID. No. 54):ATCTCGAGCTTAGATCTTTTGTTCTGGATTTCTC Primer ERG2-5′ (SEQ. ID. No. 55):CTGCGGCCGCACCATGAAGTTTTTCCCACT CC Primer ERG2-3′ (SEQ. ID. No. 56):TTCTCGAGTTAGAACTTTTTGTTTTGCAACAAG Primer ERG3-5′ (SEQ. ID. No. 57):CTGCGGCCGCAATATGGATTTGGTCTTAGAAGTCG Primer ERG3-3′ (SEQ. ID. No. 58):AACTCGAGTCAGTTGTTCTTCTTGGTATTTG Primer ERG4-5′ (SEQ. ID. No. 59):CTGCGGCCGCACTATGGCAAAGGATAATAGTGAG Primer ERG4-3′ (SEQ. ID. No. 60):TTCTCGAGCTAGAAAACATAAGGAATAAAGAC Primer Δ24R-5′ (SEQ. ID. No. 47):CTGCGGCCGCAAGATGGAGCCCGCCGTGTCGC Primer Δ24R-3′ (SEQ. ID. No. 48)AACTCGAGTCAGTGCCTTGCCGCCTTGC

1.3 Preparation of the Integration Vectors pUG6-tHMG, pUG6-ERG1,pUG6-ERG11 1.3.1 pUG6-tHMG

The DNA sequence for the expression cassette composed ofADH1-promoter-tHMG-tryptophan-terminator was isolated from the vectorYepH2 (Polakowski, T., Stahl, U., Lang, C. (1998): Overexpression of acytosolic HMG-CoA reductase in yeast leads to squalene accumulation.Appl. Microbiol. Biotechnol. 49: 66-71) by restriction with the enzymesEcoRV and Bsp681 (Nrul) by using standard methods. The DNA fragmentobtained was cloned with blunt ends into the EcoRV cleavage site of thevector pUG6 (Guldener, U et al. (1996): A new efficient gene disruptioncassette for repeated use in budding yeast, Nucleic Acids Res. July 1;24(13):2519-24), resulting in the vector denoted pUG6-tHMG (FIG. 1).

1.3.2 pUG6-ERG1

The DNA sequence for the expression cassette composed ofADH1-promoter-ERG1-tryptophan-terminator was isolated from the vectorpFlat3-ERG1 by restriction with the enzymes Nhel and Bsp681 (Nrul),using standard methods. After Klenow treatment, the DNA fragmentobtained was cloned with blunt ends into the EcoRV cleavage site of thevector pUG6 (Guldener, U et al. (1996): A new efficient gene disruptioncassette for repeated use in budding yeast, Nucleic Acids Res. July 1;24(13):2519-24), resulting in the vector denoted pUG6-ERG1 (FIG. 2).

1.3.3 pUG6-ERG11

The DNA sequence for the expression cassette composed ofADH1-promotor-ERG11-tryptophan-terminator was isolated from the vectorpFlat3-ERG11 by restriction with the enzymes EcoRV and Bsp681 (Nrul)using standard methods. The DNA fragment obtained was cloned with bluntends into the EcoRV cleavage site of the vector pUG6 (Guldener, U et al.(1996): A new efficient gene disruption cassette for repeated use inbudding yeast, Nucleic Acids Res. July 1; 24(13):2519-24), resulting inthe vector denoted pUG6-ERG11 (FIG. 3).

1.4. Integrative Transformation of the Expression Cassettes into theYeast Strains GRF or GRFura3

After plasmid isolation, fragments of the vectors pUG6-tHMG, pUG6-ERG1and pUG6-ERG11 were amplified by means of PCR in such a way that theresulting fragments consist of the following components:loxP-kanMX-loxP-ADH1 promoter-target gene-tryptophan terminator, withtarget gene meaning tHMG, ERG1 and, ERG11 and kanMX respectively,meaning a kanamycin-resistance gene.

The selected primers were oligonucleotide sequences which contain in theannealing region the sequences beyond the cassettes to be amplified ofthe vector pUG6-target gene and which contain at the 5′ and 3′protruding ends in each case 40 base pairs of the 5′ or 3′ sequence ofthe integration locus. This ensures that on the one hand the entirefragment, including KanMX and target gene, is amplified and, on theother hand, this fragment can then be transformed into yeast and beintegrated by homologous recombination into the target gene locus of theyeast. Depending on the target gene locus in the yeast, the followingoligonucleotide sequences were used as primers:

For integration at the URA3 gene locus: TABLE-US-00003 For integrationat the URA3 gene locus: URA3-Crelox-5′ (SEQ. ID. No. 33): 5′-ATGTCGAAAGCTACATATAA GGAACGTGCT GCATCTCATC CCAGCTGAAG CTTCGTACGC-3′ URA3-Crelox-3′(SEQ. ID. No. 34): 5′-TTAGTTTTGC TGGCCGCATC TTCTCAAATA TGCTTCCCAGGCATAGGCCA CTAGTGGATC TG-3′ For integration at the LEU2 gene locus:LEU2-Crelox-5′ (SEQ. ID. No. 35): 5′-GAATACTCAG GTATCGTAAG ATGCAAGAGTTCGAATCTCT CCAGCTGAAG CTTCGTACGC-3′ LEU2-Crelox-3′ (SEQ. ID. No. 36):5′-TCTACCCTAT GAACATATTC CATTTTGTAA TTTCGTGTCG GCATAGGCCA CTAGTGGATCTG-3′

For integration at the HIS3 gene locus: TABLE-US-00004 HIS3-Crelox-5′(SEQ. ID. No. 37): 5′-ATGACAGAGC AGAAACCCCT AGTAAAGCGT ATTACAAATGCCAGCTGAAG CTTCGTACGC-3′ HIS3-Crelox-3′ (SEQ. ID. No. 38): 5′-CTACATAAGAACACCTTTGG TGGAGGGAAC ATCGTTGGTA GCATAGGCCA CTAGTGGATC TG-3′

For integration at the ERG6 gene locus: TABLE-US-00005 ERG6-Crelox-5′(SEQ. ID. No. 39): 5′-ATGAGTGAAA CAGAATTGAG AAAAAGACAG GCCCAATTCACCAGCTGAAG CTTCGTACGC-3′ ERG6-Crelox-3′ (SEQ. ID. No. 40): 5′-TTATTGAGTTGCTTCTTGGG AAGTTTGGGA GGGGGTTTCG GCATAGGCCA CTAGTGGATC TG-3′

For integration at the ERG5 gene locus: TABLE-US-00006 ERG5-Crelox-5′(SEQ. ID. No. 41): 5′-ATGAGTTCTG TCGCAGAAAA TATAATACAA CATGCCACTCCCAGCTGAAG CTTCGTACGC-3′ ERG5-Crelox-3′ (SEQ. ID. No. 42): 5′-TTATTCGAAGACTTCTCCAG TAATTGGGTC TCTCTTTTTG GCATAGGCCA CTAGTGGATC TG-3′

The resistance to Geneticin (G418) served as selection marker. Theresulting strains contained a copy of the particular target gene (tHMG,ERG1 or ERG11) under the control of the ADH promoter and the tryptophanterminator. At the same time, it was possible to delete the particulargene of the target locus by integrating the expression cassette. Inorder to subsequently remove again the gene for G418 resistance, theresultant yeast strain was transformed with the crerecombinase-containing vector pSH47 (Guldener U, Heck S, Fielder T,Beinhauer J, Hegemann J H. (1996) A new efficient gene disruptioncassette for repeated use in budding yeast. Nucleic Acids Res. July 1;24(13):2519-24). This vector caused the expression of cre recombinase inthe yeast, and, as a consequence, the sequence region within the twoloxP sequences was removed by recombination, and this in turn resultedin only one of the two loxP sequences and the ADH1 promoter-targetgene-tryptophan terminator expression cassette remaining in the targetgene locus.

As a consequence, the yeast strain loses its G418 resistance again andis therefore suitable for integrating or removing further genes by meansof this “cre-lox” system into or from said yeast strain. The vectorpSH47 can then be removed selectively by cultivation on FOA medium.

Thus it is possible to integrate a plurality of target genessuccessively into the yeast strain under the control of the ADH1promoter and tryptophan terminator at various target loci.

First, a target gene is integrated at the URA3 locus or a ura3 strain isused in order to render the yeast strain uracil-auxotrophic, since thevector pSH47 contains a URA3 gene for selection of uracil-prototrophicstrains. FIG. 4 shows an example of the method.

This method produced the yeast integration and deletion strains listedin Table 1, with, in a manner known per se, the gene in lower-caseletters representing a deletion and the gene in capital lettersrepresenting an integration.

TABLE 1 Modification No. Strain name No. Strain name compared to GRFyeast strain I GRFtH1 ura3, tHMG:leu2 II GRFth1e1 ERG1:ura3, tHMG:leu2III GRFtH1E11 ura3, tHMG:leu2, ERG11:his3 IV GRFtH1E1E11 ERG1:ura3,tHMG:leu2, ERG11:his3 V GRFtH1E1E11erg5erg6 ura3, tHMG:leu2, ERG1:erg6,ERG11:erg5 VI GRFtH1erg5erg6 ura3, tHMG:leu2, erg5, erg6

The yeast strains were cultured in a culture volume of 20 ml in WMVIIImedium at 28.degree. C. and 160 rpm for 48 hours. Subsequently, 500 μlof this preculture were transferred to a 50 ml main culture of the samemedium and cultured in a baffled flask at 28.degree. C. and 160 rpm for3 days.

After 3 days, the sterols and squalene were extracted (Parks L W,Bottema C D, Rodriguez R J, Lewis T A. (1985) Yeast sterols: yeastmutants as tools for the study of sterol metabolism. Methods Enzymol.1985; 111:333-46.) and analyzed by means of gas chromatography. Thefollowing values were obtained (see Table 2).

Content of sterols 1 to 11 in [peak area/gTS] No. Strain name 1 2 3 4 56 7 8 9 10 11 I GRFtH1 9.9 0.8 0.3 1.2 1.1 1.0 0.0 0.0 0.0 0.0 4.7 IIGRFtH1E1 6.8 1.9 0.4 1.5 2.2 2.1 0.0 0.0 0.0 0.0 6.9 III GRFtH1E11 9.90.4 0.7 2.3 1.9 1.9 0.0 0.0 0.0 0.0 5.0 IV GRFtH1E1E11 6.0 1.2 0.9 3.02.3 2.2 0.0 0.0 0.0 0.0 7.2 V GRFtH1E1E11erg5erg6 5.8 0.8 0.4 23.1 0.00.0 0.0 0.0 11.8 0.0 0.0 VI GRFtH1erg5erg6 9.9 0.8 0.3 12.6 0.0 0.0 0.00.0 7.1 0.0 0.0 1 = Squalene 2 = Lanosterol 3 = Dimethylzymosterol 4 =Zymosterol 5 = Fecosterol 6 = Episterol 7 = Cholesta-7,24-dienol 8 =Cholesta-8-enol 9 = Cholesta-5,7,24 trienol 10 = 7-Dehydrocholesterol 11= Ergosterol

Example 2

Expression of the Heterologous Gene Encoding a Δ8-Δ7-Isomerase (Ebp)from Mice (Mus musculus) in Yeast

The cDNA sequence of Mus musculus Δ8-Δ7-isomerase (Moebius, F. F.,Soellner, K. E. M., Fiechter, B., Huck, C. W., Bonn, G., Glossmann, H.(1999): Histidine77, Glutamic Acid123, Threonine126, Asparagine194, andTryptophan197 of Human Emopamil Protein Are Required for in Vivo SterolΔ8-Δ7 Isomerisation. Biochem. 38, 1119-1127) was amplified by PCR fromthe cDNA clone IMAGp998A22757 (Host: E. coli DH10B) of the DeutschesResourcenzentrum fur Genomforschung [German resource center for genomeresearch] GmbH (Berlin).

The primers used here are the DNA oligomers Ebp-5′ (SEQ. ID. No. 43) andEbp-3′ (SEQ. ID. No. 44). The DNA fragment obtained was treated withrestriction enzymes Notl and Xhol and then integrated into the vectorspFlat3 and pFlat1 (FIG. 4) which likewise been treated with the enzymesNotl and Xhol beforehand by means of a ligase reaction. The resultingvectors pFlat1-EBP and pFlat3-EBP (FIG. 5 a) contain the EBP gene underthe control of the ADH promoter and the tryptophan terminator.

The expression vector pFlat3-EBP was then transformed into the yeaststrains I to VI of Table 1 from Example 1 and also into the GRFura3strain. The yeast strains obtained in this way were then cultured in aculture volume of 20 ml in WMVIII medium at 28.degree. C. and 160 rpmfor 48 hours. Subsequently, 500 μl of this preculture were transferredto a 50 ml main culture of the same medium and cultured in a baffledflask at 28.degree. C. and 160 rpm for 3 days.

The sterols were extracted after 3 days and analyzed by means of gaschromatography, as described in Example 1. The influence of theexpression of a Mus musculus Δ8-Δ7-isomerase in combination with theexpression of the transcriptionally deregulated intrinsic yeast genestHMG and/or ERG1 and/or ERG11 and/or deletion of the intrinsic yeastgenes ERG6 and ERG5 is listed in Table 3. The abbreviations have thefollowing meanings: −=decrease; 0=no change; /=not present; +, ++, +++,++++=concentrated to highly concentrated.

Influence of the genetic modifications on the sterol content compared tothe GRF yeast strain No. Strain name 1 2 3 4 5 6 7 8 9 10 11 VII GRFtH10 0 0 0 0 0 / / / / 0 pFlat3-Ebp VIIII GRFtH1E1 0 0 0 − 0 0 + / / / 0pFlat3-Ebp IX GRFtH1E11 pFlat3- 0 0 0 − 0 0 + / / / 0 Ebp X GRFtH1E1E11pFlat3- 0 0 0 − 0 0 + / / / 0 Ebp XI GRFtH1E1E11erg5erg6 0 0 0 − / / + /++ / / pFlat3-Ebp XII GRFtH1erg5erg6 0 0 0 − / / + / + / / pFlat3-Ebp 1= Squalene 2 = Lanosterol 3 = Dimethylzymosterol 4 = Zymosterol 5 =Fecosterol 6 = Episterol 7 = Cholesta-7,24-dienol 8 = Cholesta-8-enol 9= Cholesta-5,7,24 trienol 10 = 7-Dehydrocholesterol 11 = Ergosterol

Example 3

Expression of the Heterologous Gene Encoding a Δ5-Desaturase (Sc5d) fromMice (Mus musculus) in Yeast

The cDNA sequence of Mus musculus Δ5-desaturase (Nishi, S., Hideaki, N.,Ishibashi, T. (2000): cDNA cloning of the mammalian sterol C5-desaturaseand the expression in yeast mutant. Biochim. Biophys. A 1490, 106-108)was amplified by PCR from the cDNA clone IMAGp998K144618 (Host: E. coliDH10B) of the Deutsches Resourcenzentrum fur Genomforschung [Germanresource center for genome research] GmbH (Berlin). The primers usedhere are the DNA oligomers Sc5d-5′ (SEQ. ID. No. 45) and Sc5d-3′ (SEQ.ID. No. 46). The DNA fragment obtained was treated with restrictionenzymes Notl and Xhol and then integrated into the vector pFlat3 (FIG.4) which likewise had been treated with the enzymes Notl and Xholbeforehand, by means of a ligase reaction. The resulting vectorpFlat3-SC5D (FIG. 5 b) contains the SC5D gene under the control of theADH promoter and the tryptophan terminator.

The expression vector pFlat3-SC5D was then transformed into the yeaststrains I to VI of Table 1 from Example 1 and also into the GRFura3strain. The yeast strains obtained in this way were then cultured in aculture volume of 20 ml in WMVIII medium at 28.degree. C. and 160 rpmfor 48 hours. Subsequently, 500 μl of this preculture were transferredto a 50 ml main culture of the same medium and cultured in a baffledflask at 28.degree. C. and 160 rpm for 3 days.

The sterols were extracted after 3 days and analyzed by means of gaschromatography, as described in Example 1. The influence of theexpression of a Mus musculus Δ5-desaturase in combination with theexpression of the transcriptionally deregulated intrinsic yeast genestHMG and/or ERG1 and/or ERG11 and/or deletion of the intrinsic yeastgenes ERG6 and ERG5 is listed in Table 4. The abbreviations have thefollowing meanings: −=decrease; 0=no change; /=not present; +, ++, +++,++++=concentrated to highly concentrated.

TABLE 4 Influence of the genetic modifications on the sterol contentcompared to the GRF yeast strain No. Strain name 1 2 3 4 5 6 7 8 9 10 11XIII GRFtH1 pFlat3-Sc5d 0 0 0 0 0 0 / / / / 0 XIV GRFtH1E1 pFlat3-Sc5d 00 0 − 0 0 / / + / 0 XV GRFtH1E11 0 0 0 − 0 0 / / + / 0 pFlat3-Sc5d XVIGRFtH1E1E11 0 0 0 − 0 0 / / + / 0 pFlat3-Sc5d XVII GRFtH1E1E11erg5erg6 0− 0 −− / / / / +++ + / pFlat3-Sc5d XVIII GRFtH1erg5erg6 0 0 0 −− / / / /++ / / pFlat3-Sc5d 1 = Squalene 2 = Lanosterol 3 = Dimethylzymosterol 4= Zymosterol 5 = Fecosterol 6 = Episterol 7 = Cholesta-7,24-dienol 8 =Cholesta-8-enol 9 = Cholesta-5,7,24 trienol 10 = 7-Dehydrocholesterol 11= Ergosterol

Example 4

Expression of the Heterologous Gene Encoding a Δ24-Reductase (D24R) fromMice (Mus musculus) in Yeast

The cDNA sequence of Mus musculus Δ24-reductase (Waterham, H. R.,Koster, J., Romeijn, G. J., Hennekam, R. C., Vreken, P., Andersson, H.C., FitzPatrick, D. R., Kelley, R. I. and Wanders, R. J., Mutations inthe 3β-Hydroxysterol Δ24-Reductase Gene Cause Desmosterolosis, anAutosomal Recessive Disorder of Cholesterol Biosynthesis, Am. J. Hum.Genet. 69 (4), 685-694 (2001)) was amplified by PCR from the cDNA cloneIMAGp998K179532 (Host: E. coli DH10B) of the Deutsches Resourcenzentrumfur Genomforschung [German resource center for genome research] GmbH(Berlin).

The primers used here are the DNA oligomers D24R-5′ (SEQ. ID. No. 47)and D24R-3′ (SEQ. ID. No. 48). The DNA fragment obtained was treatedwith restriction enzymes Notl and Xhol and then integrated into thevector pFlat4 (FIG. 6) which likewise had been treated with the enzymesNotl and Xhol beforehand, by means of a ligase reaction. The resultingvector pFlat4-D24R (FIG. 5 d) contains the D24R gene under the controlof the ADH1 promoter and the tryptophan terminator.

The expression vector pFlat4-D24R was then transformed into the yeaststrains I to VI of Table 1 from Example 1 and also into the GRFura3strain. The yeast strains obtained in this way were then cultured in aculture volume of 20 ml in WMVIII medium at 28.degree. C. and 160 rpmfor 48 hours. Subsequently, 500 μl of this preculture were transferredto a 50 ml main culture of the same medium and cultured in a baffledflask at 28.degree. C. and 160 rpm for 3 days.

The sterols were extracted after 3 days and analyzed by means of gaschromatography, as described in Example 1. The influence of theexpression of a Mus musculus Δ24-reductase in combination with theexpression of the transcriptionally deregulated intrinsic yeast genestHMG and/or ERG1 and/or ERG11 and/or deletion of the intrinsic yeastgenes ERG6 and ERG5 is listed in Table 5. The abbreviations have thefollowing meanings: −=decrease; 0=no change; /=not present; +, ++, +++,++++=concentrated to highly concentrated.

TABLE 5 Influence of the genetic modifications on the sterol contentcompared to the GRF yeast strain No. Strain name 1 2 3 4 5 6 7 8 9 10 11XIX GRFtH1 0 0 0 0 0 0 / / / / 0 pFlat4-D24R XX GRFtH1E1 0 − − − 0 0 / // + 0 pFlat4-D24R XXI GRFtH1E11 pFlat4- 0 0 0 − 0 0 / + / + 0 D24R XXIIGRFtH1E1E11 pFlat4- 0 0 0 − 0 0 / + / + 0 D24R XXIII GRFtH1E1E11erg5erg60 − − −− / / 0 + + +++ / pFlat4-D24R XXIV GRFtH1erg5erg6 0 − − − / /0 + + ++ / pFlat4-D24R 1 = Squalene 2 = Lanosterol 3 =Dimethylzymosterol 4 = Zymosterol 5 = Fecosterol 6 = Episterol 7 =Cholesta-7,24-dienol 8 = Cholesta-8-enol 9 = Cholesta-5,7,24 trienol 10= 7-Dehydrocholesterol 11 = Ergosterol

Example 5

Coexpression of the Heterologous Genes Encoding a Δ8-Δ7-Isomerase (Ebp)from Mice (Mus musculus) and a C5-Desaturase (Sc5d) from Mice (Musmusculus) in Yeast

The expression vectors pFlat1-EBP (from Example 2) and pFlat3-SC5D (fromExample 3) were transformed into the yeast strains I to VI of Table 1 ofExample 1 and also into the GRFura3 strain. The yeast strains obtainedin this way were then cultured in a culture volume of 20 ml in WMVIIImedium at 28.degree. C. and 160 rpm for 48 hours. Subsequently, 500 μlof this preculture were transferred to a 50 ml main culture of the samemedium and cultured in a baffled flask at 28.degree. C. and 160 rpm for3 days.

The sterols were extracted after 3 days and analyzed by means of gaschromatography, as described in Example 1. The influence of theexpression of a Δ8-Δ7-isomerase and a Mus musculus C5-desaturase incombination with the expression of the transcriptionally deregulatedintrinsic yeast genes tHMG and/or ERG1 and/or ERG11 and/or deletion ofthe intrinsic yeast genes ERG6 and ERG5 is listed in Table 6. Theabbreviations have the following meanings: −=decrease; 0=no change;/=not present; +, ++, +++, ++++=concentrated to highly concentrated.

TABLE 6 Influence of the genetic modifications on the sterol contentcompared to the GRF yeast strain No. Strain name 1 2 3 4 5 6 7 8 9 10 11VVX GRFtH1 pFlat3-Ebp/ 0 0 0 − 0 0 / / + / 0 pFlat1-Sc5d XXVI GRFtH1E1pFlat3-Ebp/ 0 − 0 −− 0 0 / / + / 0 pFlat1-Sc5d XXVII GRFtH1E11 pFlat3- 00 0 −− 0 0 / / + / 0 Ebp/pFlat1-Sc5d XXVIII GRFtH1E1E11 pFlat3- 0 − − −−0 0 / / ++ / 0 Ebp/pFlat1-Sc5d XXIX GRFtH1E1E11erg5erg6 0 − 0 −− / / / /+++ + / pFlat3-Ebp/pFlat1- Sc5d XXX GRFtH1erg5erg6 0 0 0 − / / / / ++ +/ pFlat3-Ebp/pFlat1- Sc5d 1 = Squalene 2 = Lanosterol 3 =Dimethylzymosterol 4 = Zymosterol 5 = Fecosterol 6 = Episterol 7 =Cholesta-7,24-dienol 8 = Cholesta-8-enol 9 = Cholesta-5,7,24 trienol 10= 7-Dehydrocholesterol 11 = Ergosterol

Example 6

Coexpression of the Heterologous Genes Encoding a Δ8-Δ7-Isomerase (Ebp)from Mice (Mus musculus) Encoding a C5-Desaturase (Sc5d) from Mice (Musmusculus) and a Δ24-Reductase from Mice (Mus musculus) in Yeast

The expression vectors pFlat1-EBP (from Example 2) and pFlat3-SC5D (fromExample 3) and pFlat4-D24R (from Example 4) were transformed into theyeast strains I to VI of Table 1 of Example 1 and also into the GRFura3strain. The yeast strains obtained in this way were then cultured in aculture volume of 20 ml in WMVIII medium at 28.degree. C. and 160 rpmfor 48 hours. Subsequently, 500 μl of this preculture were transferredto a 50 ml main culture of the same medium and cultured in a baffledflask at 28.degree. C. and 160 rpm for 3 days.

The sterols were extracted after 3 days and analyzed by means of gaschromatography, as described in Example 1. The influence of theexpression of a Δ8-Δ7-isomerase, a Mus musculus C5-desaturase and a Musmusculus Δ24-reductase in combination with the expression of thetranscriptionally deregulated intrinsic yeast genes tHMG and/or ERG1and/or ERG11 and/or deletion of the intrinsic yeast genes ERG6 and ERG5is listed in Table 7. The abbreviations have the following meanings:−=decrease; 0=no change; /=not present; ++, +++, ++++=concentrated tohighly concentrated.

TABLE 7 Influence of the genetic modifications on the sterol contentcompared to the GRF yeast strain No. Strain name 1 2 3 4 5 6 7 8 9 10 11XXXI GRFtH1 pFlat3-Ebp/ 0 0 0 − 0 0 / / / + 0 pFlat1-Sc5d/pFlat4- D24RXXXII GRFtH1E1 pFlat3-Ebp/ 0 − 0 −− 0 0 / / / + 0 pFlat1-Sc5d/pFlat4-D24R XXXIII GRFtH1E11 pFlat3- 0 0 0 −− 0 0 / / / + 0 Ebp/pFlat1-Sc5d/pFlat4-D24R XXXIV GRFtH1E1E11 pFlat3- 0 − − −− 0 0 / / / + 0Ebp/pFlat1-Sc5d/ pFlat4-D24R XXXV GRFtH1E1E11erg5erg6 0 − 0 −−− / / // + ++++ / pFlat3-Ebp/pFlat1- Sc5d/pFlat4-D24R XXXVI GRFtH1erg5erg6 0 00 − / / / / ++ +++ / pFlat3-Ebp/pFlat1- Sc5d/pFlat4-D24R 1 = Squalene 2= Lanosterol 3 = Dimethyl zymosterol 4 = Zymosterol 5 = Fecosterol 6 =Episterol 7 = Cholesta-7,24-dienol 8 = Cholesta-8-enol 9 =Cholesta-5,7,24 trienol 10 = 7-Dehydrocholesterol 11 = Ergosterol

1. A method for preparing 7-dehydrocholesterol and/or the biosyntheticintermediates and/or secondary products thereof by culturing organismswhich, compared to the wild type, have an increased activity of at leastone of the activities selected from the group consisting ofΔ8-Δ7-isomerase activity, Δ5-desaturase activity and Δ24-reductaseactivity.
 2. The method of claim 1, wherein the organisms, compared tothe wild type, have an increased activity of at least two of theactivities selected from the group consisting of Δ8-Δ7-isomeraseactivity, Δ5-desaturase activity and Δ24-reductase activity.
 3. Themethod of claim 1, wherein the organisms, compared to the wild type,have an increased Δ8-Δ7-isomerase activity, Δ5-desaturase activity andΔ24-reductase activity.
 4. The method of claim 1, wherein theΔ8-Δ7-isomerase activity is increased by increasing, compared to thewild type, gene expression of a nucleic acid encoding a Δ8-Δ7-isomerase.5. The method of claim 4, wherein gene expression is increased byintroducing into the organism one or more nucleic acids encoding aΔ8-Δ7-isomerase.
 6. The method of claim 5, wherein nucleic acids areintroduced, which encode proteins comprising the amino acid sequenceSEQ. ID. NO. 2 or a sequence derived from this sequence by substitution,insertion or deletion of amino acids, which is at least 30% identical atthe amino acid level with the sequence SEQ. ID. NO. 2, and having theenzyme property of a Δ8-Δ7-isomerase.
 7. The method of claim 6, whichcomprises introducing a nucleic acid comprising the sequence SEQ. ID.NO.
 1. 8. The method of claim 1, wherein the Δ5-desaturase activity isincreased by increasing, compared to the wild type, gene expression of anucleic acid encoding a Δ5-desaturase.
 9. The method of claim 8, whereingene expression is increased by introducing into the organism one ormore nucleic acids encoding a Δ5-desaturase.
 10. The method of claim 9,wherein nucleic acids are introduced, which encode proteins comprisingthe amino acid sequence SEQ. ID. NO. 4 or a sequence derived from thissequence by substitution, insertion or deletion of amino acids, which isat least 30% identical at the amino acid level with the sequence SEQ.ID. NO. 4, and having the enzyme property of a Δ5-desaturase.
 11. Themethod of claim 10, which comprises introducing a nucleic acidcomprising the sequence SEQ. ID. NO.
 3. 12. The method of claim 1,wherein the Δ24-reductase activity is increased by increasing, comparedto the wild type, gene expression of a nucleic acid encoding aΔ24-reductase.
 13. The method of claim 12, wherein gene expression isincreased by introducing into the organism one or more nucleic acidsencoding a Δ24-reductase.
 14. The method of claim 13, wherein nucleicacids are introduced, which encode proteins comprising the amino acidsequence SEQ. ID. NO. 6 or a sequence derived from this sequence bysubstitution, insertion or deletion of amino acids, which is at least30% identical at the amino acid level with the sequence SEQ. ID. NO. 6,and having the enzymic property of a Δ24-reductase.
 15. The method ofclaim 14, which comprises introducing a nucleic acid comprising thesequence SEQ. ID. NO.
 5. 16. The method of claim 1, wherein theorganisms, compared to the wild type, additionally have a reducedactivity of at least one of the activities selected from the groupconsisting of C24-methyltransferase activity and Δ22-desaturaseactivity.
 17. The method of claim 16, wherein the organisms, compared tothe wild type, have a reduced C24-methyltransferase activity and areduced Δ22-desaturase activity.
 18. The method of claim 16, wherein theC24-methyltransferase activity is reduced by reducing, compared to thewild type, gene expression of a nucleic acid encoding aC24-methyltransferase.
 19. The method of claim 18, wherein an organismis used, which has no functional C24-methyltransferase gene.
 20. Themethod of claim 16, wherein the Δ22-desaturase activity is reduced byreducing, compared to the wild type, gene expression of a nucleic acidencoding a Δ22-desaturase.
 21. The method of claim 20, wherein anorganism is used, which has no functional Δ22-desaturase gene.
 22. Themethod of claim 1, wherein the organisms additionally have, compared tothe wild type, an increased activity of at least one of the activitiesselected from the group consisting of HMG-CoA-reductase activity,lanosterol C14-demethylase activity, squalene-epoxidase activity,squalene-synthetase activity and sterol-acyltransferase activity. 23.The method of claim 22, wherein the organisms additionally have,compared to the wild type, an increased lanosterol C14-demethylaseactivity and an increased HMG-CoA-reductase activity.
 24. The method ofclaim 22, wherein the lanosterol C14-demethylase activity is increasedby increasing, compared to the wild type, gene expression of a nucleicacid encoding a lanosterol C14-demethylase.
 25. The method of claim 24,wherein gene expression is increased by introducing into the organismone or more nucleic acids encoding a lanosterol C14-demethylase.
 26. Themethod of claim 25, wherein nucleic acids are introduced, which encodeproteins comprising the amino acid sequence SEQ. ID. NO. 8 or a sequencederived from this sequence by substitution, insertion or deletion ofamino acids, which is at least 30% identical at the amino acid levelwith the sequence SEQ. ID. NO. 8, and having the enzymic property of alanosterol C14-demethylase.
 27. The method of claim 26, which comprisesintroducing a nucleic acid comprising the sequence SEQ. ID. NO.
 7. 28.The method of claim 22, wherein the HMG-CoA-reductase activity isincreased by increasing, compared to the wild type, gene expression of anucleic acid encoding an HMG-CoA reductase.
 29. The method of claim 28,wherein gene expression is increased by introducing into the organism anucleic acid construct comprising a nucleic acid which encodes anHMG-CoA reductase and whose expression in said organism, in comparisonwith the wild type, is subject to a reduced regulation.
 30. The methodof claim 29, wherein the nucleic acid construct contains a promoterwhich, in comparison with the wild-type promoter, is subjected to areduced regulation in the organism.
 31. The method of claim 29, whereinthe HMG-CoA reductase-encoding nucleic acid used is a nucleic acid whoseexpression in the organism, in comparison with the orthologous nucleicacid intrinsic to said organism, is subject to a reduced regulation. 32.The method of claim 31, wherein the HMG-CoA reductase-encoding nucleicacid used is a nucleic acid which encodes the catalytic region of saidHMG-CoA reductase.
 33. The method of claim 32, wherein nucleic acids areintroduced, which encode proteins comprising the amino acid sequenceSEQ. ID. NO. 10 or a sequence derived from this sequence bysubstitution, insertion or deletion of amino acids, which is at least30% identical at the amino acid level with the sequence SEQ. ID. NO. 10,and having the enzyme property of a HMG-CoA reductase.
 34. The method ofclaim 33, which comprises introducing a nucleic acid comprising thesequence SEQ. ID. NO.
 9. 35. The method of claim 22, wherein an organismis used which, compared to the wild type, additionally has an increasedsqualene-epoxidase activity.
 36. The method of claim 35, wherein thesqualene-epoxidase activity is increased by increasing, compared to thewild type, gene expression of a nucleic acid encoding a squaleneepoxidase.
 37. The method of claim 36, wherein gene expression isincreased by introducing into the organism one or more nucleic acidsencoding a squalene epoxidase.
 38. The method of claim 37, whereinnucleic acids are introduced, which encode proteins comprising the aminoacid sequence SEQ. ID. NO. 12 or a sequence derived from this sequenceby substitution, insertion or deletion of amino acids, which is at least30% identical at the amino acid level with the sequence SEQ. ID. NO. 12,and having the enzyme property of a squalene epoxidase.
 39. The methodof claim 38, which comprises introducing a nucleic acid comprising thesequence SEQ. ID. NO.
 11. 40. The method of claim 1, wherein theorganism used is yeast.
 41. The method of claim 1, which comprisesharvesting the organism, after culturing, and then isolating7-dehydrocholesterol and/or the biosynthetic intermediates and/orsecondary products thereof from said organism.
 42. A nucleic acidconstruct, comprising at least one nucleic acid selected from the groupconsisting of nucleic acids encoding a Δ8-Δ7-isomerase, nucleic acidsencoding a Δ5-desaturase and nucleic acids encoding a Δ24-reductase,which are functionally linked with one or more regulatory signalsensuring transcription and translation in organisms.
 43. A nucleic acidconstruct of claim 42, additionally comprising at least one nucleic acidselected from the group consisting of nucleic acids encoding an HMG-CoAreductase, nucleic acids encoding a lanosterol C14-demethylase, nucleicacids encoding a squalene epoxidase, nucleic acids encoding a squalenesynthetase and nucleic acids encoding a sterol acyltransferase, whichare functionally linked with one or more regulatory signals ensuringtranscription and translation in organisms.
 44. A combination of nucleicacid constructs, which comprises at least one nucleic acid constructselected from the groups A to C A nucleic acid construct comprisingnucleic acids encoding a Δ8-Δ7-isomerase, which are functionally linkedto one or more regulatory signals ensuring transcription and translationin organisms, B nucleic acid construct comprising nucleic acids encodinga Δ5-desaturase, which are functionally linked to one or more regulatorysignals ensuring transcription and translation in organisms and Cnucleic acid construct comprising nucleic acids encoding aΔ24-reductase, which are functionally linked to one or more regulatorysignals ensuring transcription and translation in organisms, and atleast one nucleic acid construct selected from the groups D to H Dnucleic acid construct comprising nucleic acids encoding an HMG-CoAreductase, which are functionally linked to one or more regulatorysignals ensuring transcription and translation in organisms, E nucleicacid construct comprising nucleic acids encoding a lanosterolC14-demethylase, which are functionally linked to one or more regulatorysignals ensuring transcription and translation in organisms, F nucleicacid construct comprising nucleic acids encoding a squalene epoxidase,which are functionally linked to one or more regulatory signals ensuringtranscription and translation in organisms, G nucleic acid constructcomprising nucleic acids encoding a squalene synthetase, which arefunctionally linked to one or more regulatory signals ensuringtranscription and translation in organisms, H nucleic acid constructcomprising nucleic acids encoding a sterol acyltransferase, which arefunctionally linked to one or more regulatory signals ensuringtranscription and translation in organisms.
 45. The nucleic acidconstruct of claim 42, wherein the regulatory signals comprise one ormore promoters and one or more terminators, which ensure transcriptionand translation in organisms.
 46. The nucleic acid construct of claim42, wherein regulatory signals ensuring transcription and translation inyeasts are used.
 47. A genetically modified organism, wherein thegenetic modification increases at least one of the activities selectedfrom the group consisting of Δ8-Δ7-isomerase activity, Δ5-desaturaseactivity and Δ24-reductase activity, compared to a wild type.
 48. Thegenetically modified organism of claim 47, wherein the increase of atleast one of the activities is caused by an increase in gene expressionof at least one nucleic acid selected from the group consisting ofnucleic acids encoding a Δ8-Δ7-isomerase, nucleic acids encoding aΔ5-desaturase and nucleic acids encoding a Δ24-reductase, compared tothe wild type.
 49. The genetically modified organism of claim 48, whichcontains two or more nucleic acids encoding a Δ8-Δ7-isomerase and/or twoor more nucleic acids encoding a Δ5-desaturase and/or two or morenucleic acids encoding a Δ24-reductase.
 50. The genetically modifiedorganism of claim 47, wherein the genetic modification additionallyreduces at least one of the activities selected from the groupconsisting of C24-methyltransferase activity and Δ22-desaturase activitycompared to a wild type.
 51. The genetically modified organism of claim50, wherein the reduction in at least one of the activities is caused bya reduction in gene expression of at least one nucleic acid selectedfrom the group consisting of nucleic acids encoding aC24-methyltransferase and nucleic acids encoding a Δ22-desaturase,compared to the wild type.
 52. The genetically modified organism ofclaim 51, which has no functional C24-methyltransferase gene and/orΔ22-desaturase gene.
 53. The genetically modified organism of claim 47,wherein the genetic modification additionally increases at least one ofthe activities selected from the group consisting of HMG-CoA-reductaseactivity, lanosterol C14-demethylase activity, squalene-epoxidaseactivity, squalene-synthetase activity and sterol-acyltransferaseactivity, compared to a wild type.
 54. The genetically modified organismof claim 53, wherein the increase in at least one of the activities iscaused by an increase in gene expression of at least one nucleic acidselected from the group consisting of nucleic acids encoding anHMG-CoA-reductase activity, nucleic acids encoding a lanosterolC14-demethylase, nucleic acids encoding a squalene epoxidase, nucleicacids encoding a squalene synthetase and nucleic acids encoding a sterolacyltransferase, compared to the wild type.
 55. The genetically modifiedorganism of claim 54, which contains two or more nucleic acids encodingan HMG-CoA reductase and/or two or more nucleic acids encoding alanosterol C14-demethylase and/or two or more nucleic acids encoding asqualene epoxidase and/or two or more nucleic acids encoding a squalenesynthetase and/or two or more nucleic acids encoding a sterolacyltransferase.
 56. The genetically modified organism of claim 47,which, compared to the wild type, has an increased content of7-dehydrocholesterol and/or the biosynthetic intermediates and/orsecondary products thereof.
 57. The genetically modified organism ofclaim 47, wherein the organism used is yeast.
 58. The use of agenetically modified organism as claimed in claim 47 for preparing7-dehydrocholesterol and/or the biosynthetic intermediates and/orsecondary products thereof.
 59. The combination of nucleic acidconstructs of claim 44, wherein the regulatory signals comprise one ormore promoters and one or more terminators, which ensure transcriptionand translation in organisms.
 60. The combination of nucleic acidconstructs of claim 44, wherein regulatory signals ensuringtranscription and translation in yeasts are used.